Silk Powder Compaction for Production of Constructs with High Mechanical Strength and Stiffness

ABSTRACT

The present disclosure relates generally to compositions and methods for production of three-dimensional constructs with high mechanical strength and/or stiffness.

RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(c) of the U.S.Provisional Application No. 61/671,375, filed Jul. 13, 2012, the contentof which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant no. P41EB002520 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

TECHNICAL FIELD

The present disclosure relates generally to compositions and methods forproduction of three-dimensional constructs with high mechanical strengthand/or stiffness.

BACKGROUND

Silk material is produced by thousands of species of spiders and byworms from various insects such as mites, butterflies, and moths. Silksproduced by silkworms (typically Bombyx mori) and orb-weaving spidersare widely studied due to their impressive mechanical properties,environmental stability, biocompatibility, and tunable degradation. Inaddition, such silk can be modified to deliver antibiotics, drugs, andgrowth factors to enhance healing in biomedical applications. Biomedicalapplications have seen successful introduction of silks, dating to thefirst usage of silk sutures centuries ago. See, for example, Vepari, C.and Kaplan, D. L., “Silk as a Biomaterial,” Prog. Polym. Sci. 32 (2007),pp. 991-1007. However, there is no existing technologies that enableproduction of three-dimensional silk-based constructs with highmechanical strength and/or stiffness.

SUMMARY

Compositions and methods describe herein relate to fabrication of robustsilk material formats using a novel powder compaction technique. In someembodiments, the process is shown to generate a variety of constructgeometries with greatly enhanced mechanical performance over existingregenerated silk materials. The silk-based materials described hereinrange from monolithic materials (e.g., silk powder bound and fusedtogether under elevated temperature and pressure) to composite materials(e.g., silk-silk composites made from silk “matrix” and silk reinforcingphases combined into one consolidated material or part). The fabricationtechniques described herein can be extended to other protein ornon-protein based materials.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are photographs of silk construct made usinghigh-resolution acrylic die insert: the construct (left) and acrylic dieinsert (FIG. 1A) and stereomicroscope image of fine detail on silkconstruct (FIG. 1B).

FIGS. 1C and 1D are photographs of silk construct made using a coin as adie insert: the original coin (FIG. 1) and close-up silk constructexhibiting fine detail (FIG. 1D).

FIG. 2 is a schematic representation of a 100% silk shoe.

FIGS. 3-5 are schematic representations of a method for preparing partsof the 100% silk shoe.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

While the fiber form of silk is used in suture or textile-basedapplications, a solubilized form of silk fibroin is effective andversatile in creating unique three-dimensional morphologies andmaterials for applications that range beyond the traditionaltextile-based applications. In a typical protocol, 5 grams of B. morisilkworm coccons are immersed in 1 L of boiling 0.02 M Na₂CO₃ solutionfor 30 minutes. This degumming process removes a protein known assericin, which coats the silk fibroin and acts as a glue-like substance.Degummed fibers are collected and rinsed with distilled water threetimes, then air-dried. The fibers are then solubilized in 9.3 M LiBr(20% w/v) at 60° C. for 4 hours. A volume of 15 mL of this solution isthen dialyzed against 1 L of distilled water (water changes after 1, 3,6, 24, 36, and 48 hours) with a regenerated cellulose membrane (3500MWCO, Slide-A-Lyzer, Pierce, Rockford, Ill.). The solubilized proteinsolution is then centrifuged twice (9700 PRM, 20 min., 4° C.) to removeinsoluble particulates. Protein concentration is then determined bydrying a known volume of the silk solution under a hood for 12 hours andassessing the mass of the remaining solids. See, for example, Wray, L.S., Hu, X., Gallego, J., Georgakoudi, I., Omenetto, F. G., Schmidt, D.and Kaplan, D. L., “Effect of Processing on Silk-Based Biomaterials:Reproducibility and Biocompatibility,” Journal of Biomedical MaterialsResearch Part B: Applied Biomaterials 99B: 1 (2001), pp. 89-10, contentof which is incorporated herein by reference in its entirety.

Solubilized silk (also referred to as silk solution herein) can beprocessed to create a range of material formats, such as films, foams,fibers, gels, and sponges. Typically, these materials and or materialforms exhibit sort or flexible material response (e.g., low hardness,low tensile/compressive strength, and low flexural stiffness). Whilethese responses have not restricted the usage of silk in implantation orrepair applications involving soft tissue, there is a need to explorethe creation of silk materials and scaffolds that have much bettermechanical performance. For example, silk-based tissue engineeringconstructs ure being proposed for bone repair or replacement. For thisapplication, excellent strength and toughness properties are requisiteto provide structural support within the body.

Accordingly, in one aspect, the disclosure provides a method forpreparing an article of manufacture. Generally, the method comprisescompacting or consolidating a silk composition. The silk in thecomposition can be in an at least partially insoluble state. Aftercompaction, the composition can be in a solid state. After compaction,the composition can optionally be processed into a desired final shape.

In some embodiments, the silk fibroin composition is in form of apowder, i.e., the composition comprises silk particles. The silkparticles can be nanoparticles or microparticles. As used herein, theterm “particle” includes spheres; rods; shells; and prisms; and theseparticles can be part of a network or an aggregate. Without limitations,the particle can have any size from nm to millimeters. In someembodiments, the particles can have a size ranging from about 0.01 μm toabout 1000 μm, about 0.05 μm to about 500 μm, about 0.1 μm to about 250μm, about 0.25 μm to about 200 μm, or about 0.5 μm to about 100 μm.Further, the silk particle can be of any shape or form, e.g., spherical,rod, elliptical, cylindrical, capsule, or disc. In some embodiments, thesilk particle is a microparticle or a nanoparticle. As used herein, theterm “microparticle” refers to a particle having a particle size ofabout 1 nm to about 1000 μm. As used herein, the term “nanoparticle”refers to particle having a particle size of about 0.1 nm to about 1000nm.

Without wishing to be bound by a theory, particle size can greatlydetermine microscopic and macroscopic properties of the final product.Particle size is dependent on a number of process parameters, including,but not limited to, the size of the ceramic balls used, the amount ofsilk placed in each ball mill cup, the rotational speed (RPM) of themachine, and the duration of ball milling. Particle size in the powdercan be predicted based on some of these process parameters, e.g., withmathematical modeling and or experimentation to determine thecorrelation. For example, this can be done by milling a given volume ofsilk fibroin for varying ball mill speeds and duration. ScanningElectron Microscopy (SEM) can be performed on representative samplesfrom each experiment to determine particle size. Additional tests can berun on each sample to determine the effect of process parameters on thecolor, molecular weight, viscosity in a solution, and solubility inwater of the resulting constructs.

In some embodiments, the silk particles comprise silk fibroinsubstantially free of sericin. In some embodiments, the silk particlescomprise non-degummed silk or partially degummed silk (i.e., silk havingsome amount of sericin). Silk fibers are composed of fibroin and sericinproteins. For biomedical applications, the sericin can be removed beforeimplantation to prevent immunogenic responses. This can be done througha process known as degumming. For example, 5 grams of B. mori silkwormcocoons can be immersed in 1 L of boiling 0.02 M Na₂CO₃ solution for 30minutes. Degummed fibers can be collected and rinsed with distilledwater (e.g., three times) and air-dried. A calibrated inspection toolcan be developed to measure sericin. In some embodiments, a tunableamount of sericin can be left in the silk material after degumming. Thiscan eliminate the need to mix together silk particles prepared fromdegummed and non-degummed silk fibroin.

Silk powder can be useful in many applications, for example, as a fillerin silk gels or other silk forms or possibly as a crystallite-likematerial to enhance (acting as a catalyst) conversion of a silk solutioninto a hydrogel. For the hard silk material described in the presentprocess, in some embodiments, a liquid binder can be added to the silkparticle composition. Taking advantage of the ability of sericin proteinto act as a glue-like binder in silkworm cocoons, in some embodiments,the method disclosed herein comprises mixing in a combination of silkparticles made from degummed and silk particles made from non-degummedsilk fibroin. To control the amount of sericin, a specific proportion ofeach can be weighed and mixed together. The particles are mixed togethervigorously to ensure that the final mixture is homogeneous.

The mixture can comprise from 100% non-degummed silk to 100% degummedsilk. Without wishing to be bound by theory, the consolidation ability,level of bonding, and strength properties of the final construct can belikely highly dependent on the sericin content. Another variation thatcan occur in this step includes the addition of other silk (andnon-silk) materials to reinforce the construct. Various compositearchitectures can be used, for example, from chopped or continuous fiberreinforcement, to the embedding of textile-like reinforcing layers. Inaddition to mechanical reinforcing phases, there are many art-recognizedadditives that can be used, each of which can affect the final productdifferently.

In some embodiments, the composition comprises a mixture of silkparticles comprising degummed silk and silk particles comprisingnon-degummed silk. Ratio of degummed silk to non-degummed silk in thecomposition can range from about 50:1 (w/w) to about 1:50 (w/w). In someembodiments, ratio of degummed silk to non-degummed silk in thecomposition can range from about 25:1 (w/w) to about 1:25 (w/w), fromabout 20:1 (w/w) to about 1:20 (w/w), from about 15:1 (w/w) to about1:15 (w/w), from about 10:1 (w/w) to about 1:10 (w/w), from about 5:1(w/w) to about 1:25 (w/w), from about 1:1 (w/w) to about 1:20, fromabout 1:1 (w/w) to about 1:15 (w/w), or from about 1:2.5 (w/w) to about1:10 (w/w).

Various methods of producing silk particles (e.g., nanoparticles andmicroparticles) are known in the art. For example, a milling machine(e.g., a Retsch planetary ball mill) can be used to produce silk powder.Generally, the ball mill consists of either two of four sample cupsarranged around a central axis, which is geared such that each cuprotates both centrally and locally. Each ceramic cup is filled withsmall ceramic spheres. A range of sizes is available; balls with adiameter of 10 millimeters were are used for the milling operationsdescribed in the present disclosure. As the cups spin, the spheres crushmaterial in the cups to a small characteristic size. Both degummed andnon-degummed silk can be converted from pulverized material to powderform in the ball mill.

Before milling, a pulverization step can be used to break up silkfibroin in the form of whole cocoons or bave silk before introduction toa ball mill. If the cocoons are not shredded, it is possible that theball mill can take a significant amount of time to crush the cocoonsinto powder. One issue, however, can be related to the degradation(decreased molecular weight of silk fibroin) from pulverization. Testingwith SDS-PAGE (gel electrophoresis) has shown that pulverizing silkbefore degumming can degrade molecular weight significantly, whencompared to silk that was not pulverized. While this can have a negativeimpact on the final properties achievable in the silk constructs,elimination of this step may not provide a significant benefit. Withoutwishing to be bound by theory, the ball milling operation can degradethe silk material as well. In some embodiments, the milling can be usedto produce powders. In other embodiments, alternative powder formationtechniques can be used (e.g., lyophilization or flash freezing andcrushing). In other embodiments, alternative grates on the pulverizer,with larger holes, can be used. This can generate larger silk particlesizes.

Generally, for pulverization, dried silk is placed into a pulverizer,e.g., Fritsch Pulverisette 19, which “pulverizes” the silk by forcing itthrough a grate by the rotating action of a 5-bladed milling cutter. Toensure proper flow of the silk material through the pulverizer (e.g.,Pulverisette), a vacuum (e.g., an industrial vacuum) can be attached tothe outflow tube on the bottom of the grate. Pulverized silk can then becollected from the inside of the industrial vacuum. Generally, theresulting silk material is chopped and fluffy, made up of fairly shortsilk particles. Given the availability of additional grates with uniqueperforation size, silk particles of varying length can be produced.

In some embodiments, the silk particles can be produced by a polyvinylalcohol (PVA) phase separation method as described in, e.g.,International App. No. WO 2011/041395, the content of which isincorporated herein by reference in its entirety. Other methods forproducing silk fibroin particles are described, for example, in U.S.App. Pub. No. U.S. 2010/0028451 and PCT App. Pub. No.: WO 2008/118133(using lipid as a template for making silk microspheres or nanospheres),and in Wenk et al. J Control Release, Silk fibroin spheres as a platformfor controlled drug delivery, 2008; 132: 26-34 (using spraying method toproduce silk microspheres or nanospheres), content of all of which isincorporated herein by reference in its entirety.

In some embodiments, silk particles can be produced using afreeze-drying method as described in U.S. Provisional Application Ser.No. 61/719,146, filed Oct. 26, 2012, content of which is incorporatedherein by reference in its entirety. Specifically, silk foam can beproduced by freeze-drying a silk solution. The foam then can be reducedto particles. For example, a silk solution can be cooled to atemperature at which the liquid earner transforms into a plurality ofsolid crystals or particles and removing at least some of the pluralityof solid crystals or particles to leave a porous silk material (e.g.,silk foam). After cooling, liquid carrier can be removed, at leastpartially, by sublimation, evaporation, and/or lyophilization. In someembodiments, the liquid carrier can be removed under reduced pressure.After formation, the silk fibroin foam can be subjected to grinding,cutting, crushing, or any combinations thereof to form silk particles.For example, the silk fibroin foam can be blended in a conventionalblender or milled in a ball mill to form silk particles of desired size.

The term “compacting” can be understood to mean reduce in volume and/orincrease in density. One way of compacting the silk fibroin compositioncan be by applying pressure to the composition. Accordingly, in someembodiments, the method comprises providing a silk composition, whereinthe silk fibroin can be in an at least partially insoluble state; andapplying pressure to the composition.

The pressure can be applied using a press, e.g., designed specificallyfor this purpose. In one non-limiting example, the press is composed of4 parts—a base plate, a cavity plate, a top plate, and a piston. Thebase plate is attached to the cavity plate by four ¼″-20 bolts to form awell. The silk composition is deposited in the well, and the piston isinserted into position. The piston is machined to just fit inside thewell to minimize the amount the composition that can leak out uponcompaction. Next the top plate is bolted onto the cavity plate, and thebolls are tightened using a torque wrench such that there is a specificamount of pressure on the material inside the press. It is important forthe pressure to be sufficient and for the consistency of the compound tobe correct, otherwise the compound can leak, or the resulting materialcan be inconsistent and non-homogenous.

Applying adequate pressure is desirable during the compaction process.With insufficient pressure, the final construct can be porous and easilycrack. With over-pressure, as with the addition of too much binder,e.g., water, the compound can leak out of the press, generating a finalconstruct with poor geometric stability and poor mechanical performance.Accordingly, in some embodiments, an integrated, one-piece bottomplate/cavity plate can be developed. This can prevent leakage at thebase of the well. However, removal of the final construct can becomemore difficult. Alternatively, the well and piston can be fabricatedwith draft angles, which can allow for easier construct removal.

The pressure to be applied to the composition can be a pressure of about0.05 bar, about 0.1 bar, about 0.15 bar, about 0.2 bar, about 0.25 bar,about 0.3 bar, about 0.35 bar, about 0.4 bar, about 0.45 bar, about 0.5bar, about 0.55 bar, about 0.6 bar, about 0.65 bar, about 0.7 bar, about0.75 bar or higher. For example, the pressure can be about 1 bar, 1.25bar, 1.5 bar, 1.75 bar, 2 bar, 2.25 bar, 2.5 bar, 2.75 bar, 3 bar, 3.25bar, 3.5 bar, 3.75 bar, 4 bar, 4.25 bar, 4.5 bar, 4.75 bar, 5 bar, 5.25bar, 5.5 bar, 5.75 bar, 6 bar, 7.25 bar, 7.5 bar, 7.75 bar, 8 bar, 8.25bar, 8.5 bar, 8.75 bar, 9 bar, 9.25 bar, 9.5 bar, 9.75 bar, 10 bar, orhigher. In some embodiments, the pressure is about 1 bar or higher.

It is to be noted, that the method disclosed herein differs from themethods wherein the composition is incubated under pressure but apressure is not directly applied to the composition. In the methoddisclosed herein, the silk fibroin composition is compacted by applyinga pressure directly to the composition.

As used herein the term “insoluble state” when used in reference to asilk fibroin refers to the formation of or state of being in asubstantially amorphous, primarily beta-sheet conformation. The term“formed into an insoluble state” is not intended to reflectpolymerization of silk monomers into a silk polymer. Rather, it isintended to reflect the conversion of soluble silk fibroin to a waterinsoluble state. As used herein, silk fibroin is in an “insoluble state”if it can be pelleted by centrifugation or if it cannot be dissolved byimmersion in or rinsing with water at 37° C. or less.

Without limitation, compaction can be carried out at any desiredtemperature. In some embodiments, compaction is at room temperature. Insome other embodiments, compaction is at an elevated temperature. Asused herein, the term “elevated temperature” means a temperature higherthat room temperature. Generally, the elevated temperature is atemperature higher than about 25° C. For example, the elevatedtemperature can be temperature of about 30° C. or higher, about 35° C.or higher, about 40° C. or higher, about 45° C. or higher, about 50° C.or higher, about 55° C. or higher, about 60° C. or higher, about 65° C.or higher, about 70° C. or higher, about 75° C. or higher, about 80° C.or higher, about 85° C. or higher, about 90° C. or higher, about 95° C.or higher, about 100° C. or higher, about 105° C. or higher, about 110°C. or higher, about 115° C. or higher, about 120° C. or higher, about125° C. or higher, about 130° C. or higher, about 135° C. or higher,about 140° C. or higher, about 145° C. or higher, or about 150° C. orhigher. In some embodiments, compaction can be at room temperature,about 60° C., or about 120° C.

In some embodiments, with the composition under pressure in a compactionpress, the entire press can be placed in a preheated oven for a specificamount of time.

Without wishing to be bound by a theory, mechanistically, theconsolidation process that occurs with the silk powder is likely relatedto the glass transition temperature (Tg) of the polymer involved. Whileit is widely reported that the Tg for silk fibroin is in the range of190° C. to 210° C., the Tg can shift depending on molecular weight.Given the degradation that occurs due to the pulverizing and ballmilling operations, the silk powder generated likely has a much lowerTg. The Tg of silk before and after pulverizing and ball milling can bedetermined using analytical techniques, such as Differential ScanningCalorimetry (DSC). The temperature used during the consolidation processcan affect the mechanical property of the final construct. If thetemperature is too high or the material is left in the oven too long,sample burning can occur. If the temperature is too low or the materialis not maintained at elevated temperature long enough, the sample couldbe soft and not fully dry, leading to construct deformation,inhomogeneity, and poor mechanical robustness.

Without limitation, compaction can be for any desired period of time.For example, the compaction can be for a period of minutes, hours, ordays. For example, the compaction can be for a period of about one hour,two hours, three hours, four hours, five hours, six hours, twelve hours,one day, two days, three days or longer.

The compaction time and/or temperature can affect the sample greatly.For example, if the temperature is too low or the heating time tooshort, the sample typically does not consolidate well (not cookedthrough). If the temperature is too high or the heating time too long,the sample appears to overheat and even burn (over-cooking). In eithercase, the resulting construct can have poor geometric stability andlimited mechanical robustness.

If the compaction is at an elevated temperature, it can be desirable tocool the compacted composition before removal from removing it from thepress. Cooling (e.g., complete cooling) can be desirable before removalof the compacted composition from the press or the compacted compositioncan warp as it cools outside of the press. The compacted composition canbe cooled for any desired period of time before removal from the press.In some embodiments, the press can be removed from the oven and placedin a fume hood to cool by convection with room temperature air. Oncecompletely cool, the bolts can be released and the sample removed.

In some embodiments, the silk composition can further comprise a binder.As used herein, the term “binder” includes any additive which impartscohesive qualities and is used for the purpose of binding or holdingtogether powdered components in a solid compacted form. Suitable bindersdepend on the individual application and are known to, and can bedetermined by, the person skilled in the art. Without wishing to bebound by theory, hydration of the sericin and possibly fibroin can causethe material to become slightly sticky; e.g., recapitulating theglue-like response of sericin naturally produced by silkworms.

In some embodiments, the binders contemplated are liquids, e.g., water,salt solutions, and the like. Amount of liquid binder in the silkcomposition can range from about 0.1% (w/w) to about 75% (w/w) of thetotal of the composition. In some embodiments, amount of the liquidbinder in the silk composition can range from about 5% (w/w) to about65% (w/w) from about 10% (w/w) to about 60% (w/w), from about 15% (w/w)to about 50% (w/w), from about 20% (w/w) to about 45% (w/w), or fromabout 25% (w/w) to about 40% (w/w). In some embodiments, it is can bedesirable to use a ratio of 3 to 6 grams of silk particles for every 2ml of liquid binder. Generally, the amount of the liquid binder in thecomposition is sufficient to provide a silk composition of a desiredviscosity.

In some embodiments, the binder is a solubilized silk solution. Giventhe ability to easily adjust concentration (silk fibroin-to-waterratio), this provides additional flexibility for preparing the silkcomposition comprising the binder. Silk solution can act as a goodbinder for other forms of silk. There can be a number of potentialbenefits, beyond improved mechanical performance. The concentration,viscosity, molecular weight, and conformational makeup of the silkfibroin/water solution likely can have effects on the consistency andproperties of the material during the process and the final constructs.

The consistency of the liquid binder comprising composition needs to becorrect. With an insufficient quantity of liquid binder, the compactedcomposition can likely become inhomogeneous and possibly develop cracksand exhibit poor mechanical properties. With too much binder, thecomposition viscosity can likely become too low and prevent properconsolidation in the press (leakage from under the piston and likelydevelopment of voids or geometrical unstable constructs.

In some embodiments, the silk composition has a paste (or paste-like)consistency. In some embodiments, paste (or paste-like) consistencymeans that the composition is malleable or moldable. Paste consistencycan be stated in terms of the viscosity of the solution. In someembodiments, viscosity of the composition can range from about 0.1 toabout 250 Pa·s, from about 0.2 to about 150 Pa·s, from about 0.3 toabout 100 Pa·s, from about 0.4 to about 50 Pa·s, or from about 0.5 toabout 25 Pa·s. Compositions with overly high viscosity can be difficultto spread, smooth, and shape, while those with excessively low viscositycan be difficult to handle for molding purposes. Without wishing to bebound by a theory, compositions of higher viscosity can be used withouta mold. For example, a composition of higher viscosity can be formedinto a simple geometric shape by mechanical means, e.g. hands.Compositions of lower viscosity can be used for injection molding intomolds of predetermined shape or into molds of simple geometric shapes.Compositions of higher viscosity can also be used for injection moldinginto predetermined shapes or simple geometric shapes.

Viscosity can be measured with various types of viscometers andrheometers. A rheometer is generally used for those fluids which cannotbe defined by a single value of viscosity and therefore require moreparameters to be set and measured than is the case for a viscometer. Insome embodiments, viscosity can be determined at room temperature.

In some embodiments, a small amount of distilled water is measured andadded to the silk composition comprising silk particles, e.g., with a 1ml syringe. For example, a few drops of water can be added at a time andmixed with the silk particles. Once all water is added, a thoroughmixing yields a viscous and sticky compound that has the consistency ofsmooth peanut butter.

In some embodiments, the compacted composition is a hard material, witha ceramic-like feel. Mechanical response varies widely depending on theparameters selected throughout the process.

After compaction, the compacted composition can be processed into thefinal desired shape to obtain an article of manufacture. As used herein,the term “processing” with reference to processing into the desiredshape should be understood to include any method or process used toprovide the final shape of the manufactured article. Without limitation,such processing can include, but is not limited to, mechanical andchemical means. For example, processing can be selected from the groupconsisting of machining, turning (lathe), rolling, thread rolling,drilling, milling, sanding, punching, die cutting, blanking, broaching,extruding, chemical etching, and any combinations thereof. As usedherein, the term “machining” should be understood to include all typesof machining operations including, but run limited to, CNC machining,cutting, milling, turning, drilling, shaping, planing, broaching,sawing, burnishing, grinding, and the like. One or more of theprocessing methods can be used in combination to obtain more complex,intricate geometries. The term “machinable” means a material which canbe readily subjected to machining.

Accordingly, in some embodiments, the method comprises: (i) providing acomposition comprising silk particles; (ii) compacting the compositionby application of pressure; and (iii) processing the compactedcomposition to a desired shape.

In some embodiments, the composition is in a mold. As used herein, theterm “mold” is intended to encompass any mold, container or substratecapable of shaping, holding or supporting the silk composition. Thus,the mold in its simplest form could simply comprise a supportingsurface. The mold can be of any desired shape, and can be fabricatedfrom any suitable material including polymers (such as polysulphone,polypropylene, polyethylene), metals (such as stainless steel, titanium,cobalt chrome), ceramics (such as alumina, zirconia), glass ceramics,and glasses (such as borosilicate glass). In some embodiments, the moldcan provide a scaffold of simple geometry, which can be processed intothe final desired shape, i.e., the mold can be used to provide a blankwhich can be processed to the final shape.

As used herein, the term “silk fibroin” or “fibroin” includes silkwormfibroin and insect or spider silk proiein. See e.g., Lucas et al., 13Adv. Protein Chem. 107 (1958). Any type of silk fibroin can be usedaccording to aspects of the present invention. Silk fibroin produced bysilkworms, such as Bombyx mori, is the most common and represents anearth-friendly, renewable resource. For instance, silk fibroin used incan be attained by extracting sericin from the cocoons of B. mori.Organic silkworm cocoons are also commercially available. There are manydifferent silks, however, including spider silk (e.g., obtained fromNephila clavipes), transgenic silks, genetically engineered silks(recombinant silk), such as silks from bacteria, yeast, mammalian cells,transgenic animals, or transgenic plants, and variants thereof, that canbe used. See for example, WO 97/08315 and U.S. Pat. No. 5,245,012,content of both of which is incorporated heroin by reference in itsentirety. In some embodiments, silk fibroin can be derived from othersources such as spiders, other silkworms, bees, and bioengineeredvariants thereof. In some embodiments, silk fibroin can be extractedfrom a gland of silkworm or transgenic silkworms. See for example,WO2007/098951, content of which is incorporated herein by reference inits entirety. In some embodiments, silk fibroin is free, or essentiallyfree of sericin, i.e., silk fibroin is a substantially sericin-depletedsilk fibroin.

Degummed silk can be prepared by any conventional method known to oneskilled in the art. For example, B. mori cocoons are boiled for about upto 60 minutes, generally about 30 minutes, in an aqueous solution. Inone embodiment, the aqueous solution is about 0.02M Na₂CO₃. The cocoonsare rinsed, for example, with water to extract the sericin proteins. Thedegummed silk can be dried and used for preparing silk powder.Alternatively, the extracted silk can dissolved in an aqueous saltsolution. Salts useful for this purpose include lithium bromide, lithiumthiocyanate, calcium nitrate or other chemicals capable of solubilizingsilk. In some embodiments, the extracted silk can dissolved in about8M-12 M LiBr solution. The salt is consequently removed using, forexample, dialysis.

If necessary, the solution can then be concentrated using, for example,dialysis against a hygroscopic polymer, for example, PEG, a polyethyleneoxide, amylose or sericin. In some embodiments, the PEG is of amolecular weight of 8,000-10,000 g/mol and has a concentration of about10% to about 50% (w/v). A slide-a-lyzer dialysis cassette (Pierce, MW CO3500) can be used. However, any dialysis system can be used. Thedialysis can be performed for a time period sufficient to result in afinal concentration of aqueous silk solution between about 10% to about30%. In most cases dialysis for 2-12 hours can be sufficient. See, forexample, International Patent Application Publication No. WO2005/012606, the content of which is incorporated herein by reference inits entirety.

The silk fibroin solution can be produced using organic solvents. Suchmethods have been described, for example, in Li, M., et al., J. Appl.Poly Sci. 2001, 79, 2192-2199; Min, S., et al., Sen'l Gakkaishi 1997,54, 85-92; Nazarov, R. et al., Biomacromolecules 2004 May-June;5(3):718-26, content of all which is incorporated herein by reference intheir entirety. An exemplary organic solvent that can be used to producea silk solution includes, but is not limited to, hexafluoroisopropanol(HFIP). See, for example, International Application No. WO2004/000915,content of which is incorporated herein by reference in its entirety. Insome embodiments, the silk solution is free or essentially free oforganic solvents, i.e., solvents other than water.

Generally, any amount of silk fibroin can be present in the solution.For example, amount of silk in the solution or the composition preparedtherefrom can be from about 1% (w/v) to about 50% (w/v) of silk, e.g.,silk fibroin. In some embodiments, the amount of silk in the solution orthe composition prepared therefrom can be from about 1% (w/v) to about35% (w/v), from about 1% (w/v) to about 30% (w/v), from about 1% (w/v)to about 25% (w/v), from about 1% (w/v) to about 20% (w/v), from about1% (w/v) to about 15% (w/v), from about 1% (w/v) to about 10% (w/v),from about 5% (w/v) to about 25% (w/v), from about 5% (w/v) to about 20%(w/v), from about 5% (w/v) to about 15% (w/v). In some embodiments, thesilk in the silk solution is about 25% (w/v). In some embodiments, thesilk in the silk solution is about 6% (w/v) to about 8% (w/v). Exactamount of silk in the silk solution can be determined by drying a knownamount of the silk solution and measuring the mass of the residue tocalculate the solution concentration.

The silk fibroin can be used to fabricate a silk fibroin-based scaffoldwhich can then be used to produce silk particles for use in thedisclosed method. For example, the silk fibroin solution can be formedinto silk fibroin-based scaffold such as a fiber, film, gel, hydrogel,foam, mesh, mat, or non-woven mat. The silk fibroin-based scaffold(e.g., fiber, film, gel, hydrogel, foam, mesh, mat, or non-woven mat)can be processed by subjecting the silk fibroin-bused scaffold tomilling, grinding, cutting, crushing, or any combinations thereof toform silk particles. For example, the silk fibroin-based scaffold can beblended in a conventional blender or milled in a ball mill to form silkparticles of desired size.

The silk fibroin-based scaffold can be in any form, shape or size.Accordingly, in some embodiments, the silk fibroin-based material is inthe form of a fiber. As used herein, the term “fiber” means a relativelyflexible, unit of matter having a high ratio of length to width acrossits cross-sectional perpendicular to its length. Methods for preparingsilk fibroin fibers are well known in the art. A fiber can be preparedby electrospinning a silk solution, drawing a silk solution, and thelike. Electrospun silk materials, such as fibers, and methods forpreparing the same are described, for example in WO2011/008842, contentof which is incorporated herein by reference in its entirety.Micron-sized silk fibers (e.g., 10-600 μm in size) and methods forpreparing the same are described, for example in Mandal et al., PNAS,2012, doi: 10.1073/pnas.1119474109; U.S. Provisional Application No.61/621,209, filed Apr. 6, 2012, and PCT application no. PCT/US13/35389,filed Apr. 5, 2013, content of all of which is incorporated herein byreference

In some embodiments, the silk fibroin-based scaffold can be in the formof a film, e.g., a silk film. As used herein, the term “film” refers toa flat or tubular flexible structure. It is to be noted that the term“film” is used in a generic sense to include a web, film, sheet,laminate, or the like. In some embodiments, the film is a patternedfilm, e.g., nanopatterned film. Exemplary methods for preparing silkfibroin films are described in, for example, WO 2004/000915 and WO2005/012606, content of both of which is incorporated herein byreference in its entirety.

In some embodiments, the silk fibroin-based scaffold can be in the formof a gel or hydrogel. The term “hydrogel” is used herein to mean asilk-based material which exhibits the ability to swell in water and toretain a significant portion of water within its structure withoutdissolution. Methods for preparing silk fibroin gels and hydrogels arewell known in the art. Methods for preparing silk fibroin gels andhydrogels include, but are not limited to, sonication, vortexing, pHtitration, exposure to electric field, solvent immersion, waterannealing, water vapor annealing, and the like. Exemplary methods forpreparing silk fibroin gels and hydrogels are described in, for example,WO 2005/012606, content of which is incorporated herein by reference inits entirety. In some embodiments, the silk fibroin-based scaffold canbe in the form of a sponge or foam. Methods for preparing silk fibroingels and hydrogels are well known in the art. In some embodiments, thefoam or sponge is a patterned foam or sponge, e.g., nanopatterned foamor sponge. Exemplary methods for preparing silk foams and sponges aredescribed in, for example, WO 2004/000915, WO 2004/000255, and WO2005/012606, content of all of which is incorporated herein by referencein its entirety.

In some embodiments, the silk fibroin-based scaffold can be in the formof a cylindrical matrix, e.g., a silk tube. The silk tubes can be madeusing any method known in the art. For example, tubes can be made usingmolding, dipping, electrospinning, gel spinning, and the like. Gelspinning is described in Lovett et al. (Biomaterials, 29(35):4650-4657(2008)) and the construction of gel-spun silk tubes is described in PCTapplication no. PCT/US2009/039870, filed Apr. 8, 2009, content of bothof which is incorporated herein by reference in their entirety.Construction of silk tubes using the dip-coating method is described inPCT application no. PCT/US2008/072742, filed Aug. 11, 2008, content ofwhich is incorporated herein by reference in its entirety. Constructionof silk fibroin tubes using the film-spinning method is described in PCTapplication No. PCT/US2013/030206, filed Mar. 11, 2013 and U.S.Provisional application No. 61/613,185, filed Mar. 20, 2012.

In some embodiments, the silk fibroin-based scaffold can be porous. Forexample, the silk fibroin-matrix can have a porosity of at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, or higher. As used herein, the term “porosity”is a measure of void spaces in a material and is a fraction of volume ofvoids over the total volume, as a percentage between 0 and 100% (orbetween 0 and 1). Determination of porosity is well known to a skilledartisan, e.g., using standardized techniques, such as mercuryporosimetry and gas adsorption, e.g., nitrogen adsorption.

The porous silk-based scaffold can have any pore size. As used herein,the term “pore size” refers to a diameter or an effective diameter ofthe cross-sections of the pores. The term “pore size” can also refer toan average diameter or an average effective diameter of thecross-sections of the pores, based on the measurements of a plurality ofpores. The effective diameter of a cross-section that is not circularequals the diameter of a circular cross-section that has the samecross-sectional area as that of the non-circular cross-section.

Methods for forming pores in silk fibroin-based scaffolds are known inthe art and include, but are not limited, porogen-leaching methods,freeze-drying methods, and/or gas-forming method. Exemplary methods forforming pores in a silk-based material are described, for example, inU.S. Pat. App. Pub. Nos.: US 2010/0279112 and US 2010/0279112; U.S. Pat.No. 7,842,780; and WO2004062697, content of all of which is incorporatedherein by reference in its entirety.

Though not meant to be bound by a theory, silk fibroin-based scaffold'sporosity, structure, and mechanical properties can be controlled viadifferent post-spinning processes such as vapor annealing, heattreatment, alcohol treatment, air-drying, lyophilization and the like.Additionally, any desirable release rates, profiles or kinetics of amolecule encapsulated in the matrix can be controlled by varyingprocessing parameters, such as matrix thickness, silk molecular weight,concentration of silk in the matrix, beta-sheet conformation structures,silk II beta-sheet crystallinity, or porosity and pore sizes.

In some embodiments, the method further comprises inducing aconformational change in silk fibroin to make the silk fibroin at leastpartially insoluble. Without wishing to be bound by a theory, theinduced conformational change alters the crystallinity of the silkfibroin, e.g., Silk II beta-sheet crystallinity. The conformationalchange can be induced by any methods known in the art, including, butnot limited to, alcohol immersion (e.g., ethanol, methanol), waterannealing, shear stress, ultrasound (e.g., by sonication), pH reduction(e.g., pH titration and/or exposure to an electric field) and anycombinations thereof. For example, the conformational change can beinduced by one or more methods, including but not limited to, controlledslow drying (Lu et al., 10 Macromolecules 1032 (2009)); water annealing(Jin et al., Water-Stable Silk Films with Reduced β-Sheet Content, 15Adv. Funct. Mats. 1241 (2005); Hu et al. Regulation of Silk MaterialStructure by Temperature-Controlled Water Vapor Annealing, 12Biomacromolecules 1686 (2011)); stretching (Demura & Asakura,Immobilization of glucose oxidase with Bombyx mori silk fibroin by onlystretching treatment and its application to glucose sensor, 33 Biotech &Bioengin. 598 (1989)); compressing; solvent immersion, includingmethanol (Hofmann et al., Silk fibroin as an organic polymer forcontrolled drug delivery, 111 J Control Release. 219 (2006)), ethanol(Miyairi et al., Properties of b-glucosidase immobilized in sericinmembrane. 56 J. Fermen. Tech. 303 (1978)), glutaraldehyde (Acharya etal., Performance evaluation of a silk protein-based matrix for theenzymatic conversion of tyrosine to L-DOPA. 3 Biotechnol J. 226 (2008)),and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) (Bayraktar etal., Silk fibroin as a novel coating material for controlled release oftheophylline. 60 Eur J Pharm Biopharm. 373 (2005)); pH adjustment, e.g.,pH titration and/or exposure to an electric field (see, e.g., U.S.Patent App. No. US2011/0171239); heat treatment; shear stress (see,e.g., International App. No.: WO 2011/005381), ultrasound, e.g.,sonication (see, e.g., U.S. Patent Application Publication No. U.S.2010/0178304 and International App. No. WO2008/150861); and anycombinations thereof. Content of all of the references listed above isincorporated herein by reference in their entirety.

In some embodiments, the conformation of the silk fibroin can be alteredby water annealing. Without wishing to be bound by a theory, it isbelieved that physical temperature-controlled water vapor annealing(TCWVA) provides a simple and effective method to obtain refined controlof the molecular structure of silk biomaterials. The silk materials canbe prepared with control of crystallinity, from a low content usingconditions at 4° C. (α helix dominated silk I structure), to highestcontent of ˜60% crystallinity at 100° C. (β-sheet dominated silk IIstructure). This physical approach covers the range of structurespreviously reported to govern crystallization during the fabrication ofsilk materials, yet offers a simpler, green chemistry, approach withtight control of reproducibility. Temperature controlled water vaporannealing is described, for example, in Hu et al., Rergulation of SilkMaterial Structure By Temperature Controlled Water Vapor Annealing,Biomacromolecules, 2011, 12(5): 1686-1696, content of which isincorporated herein by reference in its entirety.

In some embodiments, alteration in the conformation of the silk fibroincan be induced by immersing in alcohol, e.g., methanol, ethanol, etc.The alcohol concentration can be at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90% or 100%. In some embodiment, alcohol concentration is100%. If the alteration in the conformation is by immersing in asolvent, the silk composition can be washed, e.g., with solvent-watergradient to remove any of the residual solvent that is used for theimmersion. The washing can be repeated one, e.g., one, two, three, four,five, or more times.

Alternatively, the alteration in the conformation of the silk fibroincan be induced with sheer stress. The sheer stress can be applied, forexample, by passing the silk composition through a needle. Other methodsof inducing conformational changes include applying an electric field,applying pressure, or changing the salt concentration.

The treatment time for inducing the conformational change can be anyperiod of time to provide a desired silk II (beta-sheet crystallinity)content. In some embodiments, the treatment time can range from about 1hour to about 12 hours, from about 1 hour to about 6 hours, from about 1hour to about 5 hours, from about 1 hour to about 4 hours, or from about1 hour to about 3 hours. In some embodiments, the sintering time canrange from about 2 hours to about 4 hours or from 2.5 horus to about 3.5hours.

When inducing the conformational change is by solvent immersion,treatment time can range from minutes to hours. For example, immersionin the solvent can be for a period of at least about 15 minutes, atleast about 30 minutes, at least about 1 hour, at least about 2 hours,at least 3 hours, at least about 6 hours, at least about 18 hours, atleast about 12 hours, at least about 1 day, at least about 2 days, atleast about 3 days, at least about 4 days, at least about 5 days, atleast about 6 days, at least about 7 days, at least about 8 days, atleast about 9 days, at least about 10 days, at least about 11 days, atleast about 12 days, at least about 13 days, or at least about 14 days.In some embodiments, immersion in the solvent can be for a period ofabout 12 hours to about seven days, about 1 day to about 6 days, about 2to about 5 days, or about 3 to about 4 days.

After the treatment to induce the conformutionul change, silk fibroincan comprise a silk II beta-sheet crystallinity content of at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, or at least about 95% butnot 100% (i.e., all the silk is present in a silk II beta-sheetconformation). In some embodiments, silk is present completely in a silkII beta-sheet conformation, i.e., 100% silk II beta-sheet crystallinity.

In some embodiments, the silk composition for compaction can compriseone or more (e.g., one, two, three, four, five or more) additives.Without wishing to be bound by a theory additive can provide one or moredesirable properties to an article of manufacture, e.g., strength,flexibility, case of processing and handling, biocompatibility,bioresorability, lack of air bubbles, surface morphology, and the like,prepared from the compacted composition. The additive can be covalentlyor non-covalently linked with silk and can be integrated homogenously orheterogeneously within the silk composition.

An additive can be selected from small organic or inorganic molecules;saccharines; oligosaccharides; polysaccharides; biologicalmacromolecules, e.g., peptides, proteins, and peptide analogs andderivatives; peptidomimetics; antibodies and antigen binding fragmentsthereof; nucleic acids; nucleic acid analogs and derivatives; glycogensor other sugars; immunogens; antigens; an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal cells; animaltissues; naturally occurring or synthetic compositions; and anycombinations thereof. Furthermore, the additive can be in any physicalform. For example, the additive can be in the form of a particle, afiber, a film, a gel, a mesh, a mat, a non-woven mat, a powder, aliquid, or any combinations thereof. In some embodiments, the additiveis a particle.

Total amount of additives in the composition can be from about 0.1 wt %to about 99 wt %, from about 0.1 wt % to about 70 wt %, from about 5 wt% to about 60 wt %, from about 10 wt % to about 50 wt %, from about 15wt % to about 45 wt %, or from about 20 wt % to about 40 wt %, of thetotal silk composition. In some embodiments, ratio of silk to additivein the composition can range from about 50:1 (w/w) to about 1:50 (w/w),from about 25:1 (w/w) to about 1:25 (w/w), from about 20:1 (w/w) toabout 1:20 (w/w), from about 10:1 (w/w) to about 1:10 (w/w), or fromabout 5:1 (w/w) to about 1:5 (w/w).

In some embodiments, the additive is a calcium phosphate material (CaP).As used herein, the term “calcium phosphate material” refers to anymaterial composed of calcium and phosphate ions. The term “calciumphosphate material” is intended to include naturally occurring andsynthetic materials composed of calcium and phosphate ions. The ratio ofcalcium to phosphate ions in the calcium phosphate materials ispreferably selected such that the resulting material is able to performits intended function. For convenience, the calcium to phosphate ionratio is abbreviated as the “Ca/P ratio.” In some embodiments, the Ca/Pratio can range from about 1:1 to about 1.67 to 1. In some embodiments,the calcium phosphate material can be calcium deficient. By “calciumdeficient” is meant a calcium phosphate material with a calcium tophosphate ratio of less than about 1.6 as compared to the idealstoichiometric value of approximately 1.67 for hydroxyapatite

The calcium phosphate material can be in the form of particles. Withoutlimitations, the calcium phosphate material particles can be of anydesired size. In some embodiments, the calcium phosphate materialparticles can have a size ranging from about 0.01 μm to about 1000 μm,about 0.05 μm to about 500 μm, about 0.1 μm to about 250 μm, about 0.25μm to about 200 μm, or about 0.5 μm to about 100 μm. Further, thecalcium phosphate material particle can be of any shape or form, e.g.,spherical, rod, elliptical, cylindrical, capsule, or disc.

In some embodiments, the calcium phosphate material particle is amicroparticle or a nanoparticle. In some embodiments, the calciumphosphate material particle has a particle size of about 0.01 μm toabout 1000 μm, about 0.05 μm to about 750 μm, about 0.1 μm to about 500μm, about 0.25 μm to about 250 μm, or about 0.5 μm to about 100 μm. Insome embodiments, the silk particle has a particle size of about 0.1 nmto about 1000 nm, about 0.5 nm to about 500 nm, about 1 nm to about 250nmm, about 10 nm to about 150 nm, or about 15 nm to about 100 nm.

The calcium phosphate material can be selected, for example, from one ormore of brushite, octacalcium phosphate, tricalcium phosphate (alsoreferred to as tricalcic phosphate and calcium orthophosphate), calciumhydrogen phosphate, calcium dihydrogen phosphate, apatite, and/orhydroxyapatite. Further, tricalcium phosphate (TCP) can be in the alphaor the beta crystal form. In some embodiments, the calcium phosphatematerial is beta-tricalcium phosphate or apatite, e.g., hydroxyapatite(HA).

The amount of the calcium phosphate material in the silk composition canrange from about 1% to about 99% (w/w or w/v). In some embodiments, theamount of the calcium phosphate material in the silk composition can befrom about 5% to about 95% (w/w or w/v), from about 10% to about 90%(w/w or w/v), from about 15% to about 80% (w/w or w/v), from about 20%to about 75% (w/w or w/v), from about 25% to about 60% (w/w or w/v), orfrom about 30% to about 50% (w/w or w/v). In some embodiments, theamount of the calcium phosphate material in the silk composition can beless than 20%.

Generally, the silk composition can comprise any ratio of silk tocalcium phosphate material. For example, the ratio of silk to calciumphosphate material in the composition can range from about 1000:1 toabout 1:1000. The ratio can be based on weight or moles. In someembodiments, the ratio of silk to calcium phosphate material in thesolution can range from about 500:1 to about 1:500 (w/w), from about250:1 to about 1:250 (w/w), from about 50:1 to about 1:200 (w/w), fromabout 10:1 to about 1:150 (w/w) or from about 5:1 to about 1:100 (w/w).

In some embodiments, the additive can be a silk-based material. Thesilk-based material can be selected from the group consisting of silkfibers, micro-sized silk fibers, unprocessed silk fibers, silkparticles, and any combinations thereof.

In some embodiments, the additive is a silk fiber. While the use of silkfibers is described in for example, US patent application publicationno. US20110046686, the previously described materials do not providemachinable silk materials as disclosed in the present disclosure.

In some embodiments, the silk fibers are microfibers or nanofibers. Insome embodiments, the additive is micron-sized silk fiber (10-600 μm).Micron-sized silk fibers can be obtained by hydrolyzing the degummedsilk fibroin or by increasing the boing time of the degumming process.Alkali hydrolysis of silk fibroin to obtain micron-sized silk fibers isdescribed for example in Mandal et al., PNAS, 2012, doi:10.1073/pnas.119474109; U.S. Provisional Application No. 61/621,209,filed Apr. 6, 2012; and PCT application no. PCT/US13/35389, filed Apr.5, 2013, content of all of which is incorporated herein by reference.Because regenerated silk fibers made from HFIP silk solutions aremechanically strong, the regenerated silk fibers can also be used asadditive.

In some embodiments, the silk fiber is an unprocessed silk fiber, e.g.,raw silk or raw silk fiber. The term “raw silk” or “raw silk fiber”refers to silk fiber that has not been treated to remove sericin, andthus encompasses, for example, silk fibers taken directly from a cocoon.Thus, by unprocessed silk fiber is meant silk fibroin, obtained directlyfrom the silk gland. When silk fibroin, obtained directly from the silkgland, is allowed to dry, the structure is referred to as silk I in thesolid state. Thus, an unprocessed silk fiber comprises silk fibroinmostly in the silk I conformation. A regenerated or processed silk fiberon the other hand comprises silk fibroin having a substantial silk II orbeta-sheet crystallinity.

Because implantation and post-surgical imaging of current resorbablefixation devices is a problem, the article of manufacture, e.g., medicaldevices such as orthopedic screws or other fasteners can be enhancedwith iron particles. Accordingly, in some embodiments, the additive isan iron particle. Without wishing to be bound by a theory, it isbelieved that the iron particles can help the surgeon duringimplantation due to a magnetic screw that can be attracted to a screwdriver head. Further, once the surgery is complete, the surgeon couldquickly check that all components are properly placed and have notmigrated or failed with a simple magnetic sensor. This would allow for afirst pass check of surgical errors and allow the surgeon to reopen thewound and fix the problem before the patient leaves the operating room.This would save on time, money, and recovery time.

In some embodiments, the additive is a biocompatible polymer. Exemplarybiocompatible polymers include, but are not limited to, a poly-lacticacid (PLA), poly-glycolic acid (PGA), poly-lactide-co-glycolide (PLGA),polyesters, poly(ortho ester), poly(phosphazine), poly(phosphate ester),polycaprolactone, gelatin, collagen, fibronectin, keratin, polyasparticacid, alginate, chitosan, chitin, hyaluronic acid, pectin,polyhydroxyalkanoates, dextrans, and polyanhydrides, polyethylene oxide(PEO), poly(ethylene glycol) (PEG), triblock copolymers, polylysine,alginate, polyaspartic acid, any derivatives thereof and anycombinations thereof. Other exemplary biocompatible polymers amenable touse according to the present disclosure include those described forexample in U.S. Pat. Nos. 6,302,848; 6,395,734; 6,127,143; 5,263,992;6,379,690; 5,015,476; 4,806,355; 6,372,244; 6,310,188; 5,093,489;387,314; 6,325,810; 6,337,198; 6,267,776; 5,576,881; 6,245,537;5,902,800; and 5,270,419, content of all of which is incorporated hereinby reference.

In some embodiments, the biocompatible polymer is PEG or PEO. As usedherein, the term “polyethylene glycol” or “PEG” means an ethylene glycolpolymer that contains about 20 to about 2000000 linked monomers,typically about 50-1000 linked monomers, usually about 100-300. PEG isalso known as polyethylene oxide (PEO) or polyoxyethylene (POE),depending on its molecular weight. Generally PEG, PEO, and POE arechemically synonymous, but historically PEG has tended to refer tooligomers and polymers with a molecular mass below 20,000 g/mol, PEO topolymers with a molecular mass above 20,000 g/mol, and POE to a polymerof any molecular mass. PEG and PEO are liquids or low-melting solids,depending on their molecular weights. PEGs are prepared bypolymerization of ethylene oxide and are commercially available over awide range of molecular weights from 300 g/mol to 10,000,000 g/mol.While PEG and PEO with different molecular weights find use in differentapplications, and have different physical properties (e.g. viscosity)due to chain length effects, their chemical properties are nearlyidentical. Different forms of PEG are also available, depending on theinitiator used for the polymerization process—the most common initiatoris a monofunctional methyl ether PEG, or methoxypoly(ethylene glycol),abbreviated mPEG. Lower-molecular-weight PEGs are also available aspurer oligomers, referred to as monodisperse, uniform, or discrete PEGsare also available with different geometries.

As used herein, the term PEG is intended to be inclusive and notexclusive. The term PEG includes poly(ethylene glycol) in any of itsforms, including alkoxy PEG, difunctional PEG, multiarmed PEG, forkedPEG, branched PEG, pendent PEG (i.e., PEG or related polymers having oneor more functional groups pendent to the polymer backbone), or PEG Withdegradable linkages therein. Further, the PEG backbone can be linear orbranched. Branched polymer backbones are generally known in the art.Typically, a branched polymer has a central branch core moiety and aplurality of linear polymer chains linked to the central brunch core.PEG is commonly used in branched forms that can be prepared by additionof ethylene oxide to various polyols, such as glycerol, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)m in which R represents thecore moiety, such as glycerol or pentaerythritol, and m represents thenumber of arms. Multi-armed PEG molecules, such as those described inU.S. Pat. No. 5,932,462, which is incorporated by reference herein inits entirety, can also be used as biocompatible polymers.

Some exemplary PEGs include, but are not limited to, PEG20, PEG30,PEG40, PEG60, PEG80, PEG100, PEG115, PEG200, PEG300, PEG400, PEG500,PEG600, PEG1000, PEG1500, PEG2000, PEG3350, PEG4000, PEG4600, PEG5000,PEG6000, PEG8000, PEG11000, PEG12000, PEG15000, PEG20000, PEG250000,PFG500000, PEG100000, PEG2000000 and the like. In some embodiments, PEGis of MW 10,000 Dalton. In some embodiments, PEG is of MW 100,000, i.e.PEO of MW 100,000.

In some embodiments, the additive is an enzyme that hydrolyzes silkfibroin. Without wishing to be bound by a theory, such enzymes can beused to control the degradation of the article of manufacture.

Article of Manufacture

Silk-based materials can be used to produce tissue scaffolds for tissueengineering applications. While these tissue scaffolds take advantage ofthe biocompatibilily, tunable degradation, and other properties of silk.In some embodiments, they typically cannot withstand the loadingconditions experienced by structural tissue (e.g., bone) in the body.Accordingly, mechanically robust silk materials are developed, and suchmaterial formats can range from monolithic to composite structures(silk-silk composites: silk reinforcing phase bound by a second silkmaterial phase).

One application area for robust monolithic and composite silk materialis in creating tissue engineering scaffolds for human tissuerepair/replacement in areas where in vivo physiological loadingconditions may be significant. For example, such material can be used toreplace the traditional metal plate and screw components used in areconstructive orthopedic surgery. Other biomedical applications includeusage as an internal fracture stabilizer (smart splint used as an in invivo brace) or void filling where bone defects or disease havecompromised mechanical stability.

Hard, strong, lightweight, and biodegradable monolithic and compositesilk materials are not limited to biomedical applications. Machinecomponents, such as nuts, bolts, and gears could potentially beconstructed of silk. Everyday consumer items, such as biodegradabledishware, plastic ware, or food containers could be silk-based. Giventhe ability to mold the silk materials described, fairly complex shapescan be created, along with the ability to emboss and imprint images,numbers, and codes. The properties of the silk material can be enhancedand specifically tailored through the addition of other material phases.For example, short or continuous silk have fiber can be incorporated ina composite construct to enhance material toughness. Optically clearfiber (including silk-based material) can be embedded to provide sensingand information transmission capability. By combining multiple silkmaterial formats, entirely unique products can be fabricated, e.g.,construction of a 100% silk apparel and/or accessories. In oneembodiment, a 100% silk shoe can be fabricated by combining multiplesilk material formats: for example, the hard monolithic and compositesilk materials can be combined with silk foams, films, and fibers tomake the desired shoe form.

Accordingly, the disclosure also provides an article of manufactureprepared by the method described herein. The article of manufactureprepared according the method described herein is biocompatible and/orat least partially bioresorbable. As used herein, the term“biocompatible” refers to a material that does not elicit a substantialimmune response in the host.

By “bioresorbable” is meant the ability of a material to be resorbed orremodeled in vivo. The resorption process involves degradation andelimination of the original implant material through the action of bodyfluids, enzymes or cells. The resorbed materials can be used by the hostin the formation of new tissue, or it can be otherwise re-utilized bythe host, or it can be excreted. The article of manufacture describedherein can have a resorption half-life of approximately 6 months toapproximately 12 months. In some embodiments, the article of manufacturehas a resorption half-life of approximately 9 months. The article ofmanufacture can be completely resorbed in approximately 12 months toapproximately 24 months. In some embodiments the material is completelyresorbed in approximately 12 months.

In some embodiments, the article of manufacture described herein hascompressive strength, compressive toughness and compressive elasticmodulus values approximate to those of healthy human bone and enablesimmediate load-bearing. Without wishing to be bound by a theory,load-bearing properties can also prevent unwanted resorption of adjacentbone resulting from high local stress concentration or stress-shielding.

Compressive toughness is the capacity of a material to resist fracturewhen subjected to axially directed pushing forces. Bu definition, thecompressive toughness of a material is the ability to absorb mechanical(or kinetic) energy up to the point of failure. Toughness is measured inunits of joules per cubic meter (Jm⁻³) and can be measured as the areaunder a stress-strain curve. In some embodiments, the article ofmanufacture described herein has a compressive toughness of about 1 kJm⁻³ to about 20 kJm⁻³ or about 1 kJm⁻³ to approximately 5 kJm⁻³ at 6%strain as measured by the J-integral method. In one embodiment, articleof manufacture has a compressive toughness of about 1.3 kJm⁻³, which isthe approximate compressive toughness of healthy bone.

Compressive strength is the capacity of a material to withstand axiallydirected pushing forces. By definition, the compressive strength of amaterial is that value of uniaxial compressive stress reached when thematerial fails completely. A stress-strain curve is a graphicalrepresentation of the relationship between stress derived from measuringthe load applied on the sample (measured in MPa) and strain derived frommeasuring the displacement as a result of compression of the sample. Theultimate compressive strength of the material can depend upon the targetsite of implantation. For example, if the material is for placement nextto osteoporotic cancellous bone, to avoid high stress accumulation andstress shielding, the material can comprise a compressive strength(stress to yield point) of approximately 0.1 MPa to approximately 2 MPa.If the material is intended for placement next to healthy cancellousbone, the material can comprise an ultimate compressive strength (stressto yield point) of approximately 5 MPa. Alternatively, if the materialis intended for placement next to cortical bone, the material cancomprise an ultimate compressive strength (stress to yield point) of atleast 40 MPa.

Generally, the article of manufacture described herein comprises anultimate compressive strength (stress to yield point) of at least 5 MPa,at least 10 MPa, at leaste 15 MPa, at least 20 MPa, at least 25 MPa, atleast 30 MPa, at least 35 MPa, at least 40 MPa, at least 45 MPa, atleast 50 MPa, at least 55 MPa, at least 60 MPa, at least 65 MPa, atleast 70 MPa, at least 75 MPa, at least 80 MPa, at least 85 MPa, atleast 90 MPa, at least 95 MPa, at least 100 MPa, at least 105 MPa, atleast 110 MPa, at least 115 MPa, at least 120 MPa, at least 125 MPa, atleast 130 MPa, at least 135 MPa, at least 140 MPa, at least 145 MPa, atleast 150 MPa, or at least 155 MPa.

For example, the article of manufacture described herein comprises anultimate compressive strength of about 5 MPa to about 140 MPa, about 20MPa to about 130 MPa, from about 60 MPa to about 125 MPa, or from about90 to about 120 MPa. In some embodiments, the article of manufacturedescribed herein comprises an ultimate compressive strength (stress toyield point) of at least 100 MPa. In one embodiment, the article ofmanufacture described herein comprises an ultimate compressive strength(stress to yield point) of approximately 104 MPa. In some embodiment,the article of manufacture described herein has a compressive strengthof from about 20 MPa to about 130 MPa at 5% strain.

Compressive elastic modulus is the mathematical description of thetendency of a material to be deformed elastically (i.e. non-permanently)when a force is applied to it. The Young's modulus (E) describes tensileelasticity, or the tendency of a material to deform along an axis whenopposing forces are applied along that axis; it is defined as the ratioof tensile stress to tensile strain (measured in MPa) and is otherwiseknown as a measure of stiffness of the material. The elastic modulus ofan object is defined as the slope of the stress-strain curve in theelastic deformation region. The article of manufacture described hereincan comprise a compressive elastic modulus of between approximately 100MPa and approximately 5,000 MPa GPa at 5% strain. In some embodiments,the article of manufacture described herein comprises a compressiveelastic modulus of between approximately 200 MPa and 750 MPa, betweenapproximately 250 MPa and 700 MPa, between approximately 300 MPa and 650MPa, between approximately 400 MPa and 600 MPa, or between approximately450 MPa and 550 MPa at 5% strain.

In some embodiments, article of manufacture described herein has a meancompressive elastic modulus of about 525 MPa. In some embodiments, thearticle of manufacture described herein can comprise a compressiveelastic modulus of at least 100 MPa, at least 150 MPa, at least 200 MPa,at least 250 MPa, at least 300 MPa, at least 350 MPa, at least 400 MPa,at least 450 MPa, at least 500 MPa, or at least 525 MPa.

Density of the article of manufacture can range from about 1 g/cm³ toabout 10 g/cm³. For example, the density can be between about 1.05 g/cm³to about 5 g/cm³, between about 1.1 g/cm³ to about 2.5 g/cm³, betweenabout 1.2 g/cm³ to about 2.0 g/cm³, between about 1.25 g/cm³ to about1.5 g/cm³. In some embodiments, density of the article of manufacture isabout 1.32 g/cm³.

The article of manufacture can be used for medical applications, e.g.medical devices, or the article can be for non-medical applications.

As used herein, the term medical device is intended to encompass alltypes of medical devices, including those used in connection with eitherexternal or internal treatment of a mammal. Medical devices used in theexternal treatment of a mammal include, but are not limited to, wounddressings, burn dressings or other skin coverings, and surgical thread.Medical devices used in the internal treatment of a mammal include, butare not limited to, vascular grafts, stents, catheters, valves,artificial joints, artificial organs, surgical thread, and the like.

Exemplary medical devices include, but are not limited to, an orthopedicimplant, a facial implant, a nasal implant (e.g., for nasalreconstruction), a suture anchor, a dental implant, a Swansonprosthetic, and any combinations thereof. In some embodiments, thearticle of manufacture is a continuous, one-phase suture anchor.

As used herein, the term “orthopedic implant” includes within its scopeany device intended to be implanted into the body of a vertebrateanimal, in particular a mammal such as a human, for preservation andrestoration of the function of the musculoskeletal system, particularlyjoints and bones, including the alleviation of pain in these structures.Exemplary orthopedic implants include, but are not limited to,orthopedic screws, orthopedic plates, orthopedic rods, orthopedictulips, or any combinations thereof.

In one embodiments, the article of manufacture is a tapping screw, e.g.,self-tapping screw.

In some embodiments, the article of manufacture is a suture anchor.Suture anchor are composed of an anchor, eyelet, and suture. The anchoris inserted to the bone which can be a screw mechanism or interferencefit and the eyelet is the hole or loop in the anchor through which thesuture passes.

As used herein, the term “dental implant” includes within its scope anydevice intended to be implanted into the oral cavity of a vertebrateanimal, in particular a mammal such as a human, in tooth restorationprocedures. Dental implants can also be denoted as dental prostheticdevices. Generally, a dental implant is composed of one or severalimplant parts. For instance, a dental implant usually comprises a dentalfixture coupled to secondary implant parts, such as an abutment and/or adental restoration such as a crown, bridge or denture. However, anydevice, such as a dental fixture, intended for implantation can alone bereferred to as an implant even if other parts are to be connectedthereto. Dental implants are presently preferred embodiments.

Bone screws consist of a thread portion and head used for insertion andstabilization of associated equipment such as bone plates.

The Swanson Finger Joint Implant is a flexible intramedullary-stemmedone-piece implant that helps restore function to hands and wristsdisabled by rheumatoid, degenerative or traumatic arthritis. It iscomposed of a silicone elastomer and its primary function is to helpmaintain proper joint space and alignment with good lateral stabilityand minimal flexion-extensional restriction. These implants bear minimalload as the majority of the compressive loads are distributed to thebones.

A nasal reconstruction is performed in order to create an aestheticallyinconspicuous nose while maintaining function. Structural grafts areoften required to provide rigidity to the sidewall and resist lateralcollapse and establish nasal contour and projection. Current materialsinclude alloplasts such as silicone and porous high density polyethyleneas well has homografts such as alloderm or rib cartilage.

Otoplasty is the process of reconstructing partial or total ear defectstypically resulting from congenital hypoplasia, trauma, cancer ablation,and prominent ears. The ears can be reconstructed by using cartilagefrom the rib cage or an artificial ear can be created. The rib cartilageis carved and wired together using fine stainless steel wire to create avery detailed framework.

In addition to the above-discussed specific medical devices andimplants, the method disclosed herein can be used for facial implants(dermal fillers, cheek implants, eye socket), occuloplasty, lipenhancement, reproductive organ plastic surgeries (penile implant,vaginaplasty, sex conversion), buttock augmentation, and other“plastys.”

Non-medical applications include manufacturing of dice, thumbtacks,bullets, children's toys (e.g., building blocks, Legos, Checkers, etc. .. . ), and biodegradable plastic alternatives.

In some embodiments, the article of manufacture described herein isosteoconductive. Osteoconductivity is generally defined as the abilityof a material to facilitate the migration of osteogenic cells to thesurfaces of a scaffold through the fibrin clot established immediatelyafter implantation the material. The porosity of a material affects theosteoconductivity of that material.

In some embodiments, the article of manufacture described herein isosteoinductive. Osteoinductivity is generally defined as the ability toinduce non-differentiated stem cells or osteoprogenitor cells(osteoblasts), which is a component of osseous (bone) tissue, todifferentiate into osteoblasts. The simplest test of osteoinductivity isthe ability to induce the formation of hone in tissue locations such asmuscle, which do not normally form bone (ectopic bone growth). It isgenerally understood that article of manufacture described herein can bemade osteoinductive by adding growth factors such as rhBMP-2(recombinant human bone morphogenic protein-2) to them. Themineralization and the addition of growth factors can affect theosteoinducivity of a material.

In some embodiments, the article of manufacture described herein isosteogenic and shows new bone formation after implantation in vivo.Osteogenesis is the process of laying down new bone material usingosteoblasts. Osteoblasts build bone by producing osteoid to form anosteoid matrix, which is composed mainly of Type I collagen. Osseoustissue comprises the osteoid matrix and minerals (mostly with calciumphosphate) that form the chemical arrangement termed calciumhydroxyapatite. Osteoblasts are typically responsible for mineralizationof the osteoid matrix to form osseous tissue. Without wishing to bebound by a theory, the osteoconductivity and osteoinductivity of thematerial has an impact on osteogenesis. The material can show new boneformation within 6 months of implantation in vivo. In some embodiments,the material shows new bone formation within 8 weeks of implantation invivo.

In some embodiments, the article of manufacture described herein cancomprise one or more supplementary material. The supplementary materialis selected based upon its compatibility with one or more components ofthe silk composition and its ability to impart properties (biological,chemical, physical, or mechanical) to the composite, which are desirablefor a particular therapeutic purpose or for post-sterilizationstability. For example, the supplementary material can be selected toimprove tensile strength and hardness, increase fracture toughness, andprovide imaging capability of the paste after implantation, hydration,and hardening. The supplementary materials are desirably biocompatible.

The supplementary material can be present in the silk composition invarying amounts and in a variety of physical forms, dependent upon theanticipated therapeutic use. For example, the supplementary material canbe in the form of solid structures, such as sponges, meshes, films,fibers, gels, filaments or particles, including microparticles andnanoparticles. The supplementary material itself can be a composite. Thesupplementary material can be a particulate or liquid additive or dopingagent.

In many instances, it is desirable that the supplementary material bebioresorbable. Bioresorbable material for use as supplementary materialinclude, without limitation, polysaccharides, nucleic acids,carbohydrates, proteins, polypeptides, poly(α-hydroxy-acids), poly(lactones), poly(amino acids), poly(anhydrides), poly (orthoesters),poly (anhydride-co-imides), poly (orthocarbonates), poly(α-hydroxyalkanoates), poly (dioxanones), and poly(phosphoesters). Preferably, thebioresorbable polymer is a naturally occurring polymer, such ascollagen, glycogen, chilin, starch, keratins, silk, demineralized bonematrix, and hyaluronic acid; or a synthetic polymer, such aspoly(L-lactide) (PLLA), poly(D,L-lactide) (PDLLA), polyglycolide (PGA),poly(lactide-co-glycolide (PLGA), poly(L-lactide-co-D,L-lactide),poly(D,L-lactide-co-trimethylene carbonate), polyhydroxybutyrate (PHB),poly(ε-caprolactone), poly(γ-valerolactone), poly(γ-butyrolactone),poly(caprolaclone), or copolymers thereof. Such polymers are known tobioerode and are suitable for use in the article of manufacturedescribed herein for bone grafts and the like. In addition,bioresorbable inorganic supplementary materials, such as compositionsincluding SiO2, Na2O, SaO, P205, Al2O3 and/a CaF2, can be used, as wellas salts, e.g., NaCl, and sugars, e.g., mannitol, and combinationsthereof.

Supplementary materials can also be selected from nonresorbable orpoorly resorbable materials. Suitable non-resorbable or poorlyresorbable materials include, without limitation, dextrans, celluloseand derivatives thereof (e.g., methylcellulose, carboxy methylcellulose,hydroxypropyl methylcellulose, and hydroxyethyl cellulose),polyethylene, polymethylmethacrylate (PMMA), carbon fibers,poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol),poly(vinylpyrrolidone), poly (ethyloxazoline), poly(ethyleneoxide)-co-poly(propylene oxide) block copolymers, poly(ethyleneterephthalate)polyamide, and lubricants, such as polymer waxes, lipidsand fatty acids.

The article of manufacture described herein is also useful for thepreparation of delivery vehicles for biologically active agents. Ingeneral, the only requirement is that the substance remain active withinmaterial during fabrication or be capable of being subsequentlyactivated or re-activated, or that the biologically active agent beadded to the material after at the time of implantation of into a hostor following fabrication of the vehicle.

Biologically active agents that can be incorporated into the article ofmanufacture described herein include, without limitation, organicmolecules, inorganic materials, proteins, peptides, nucleic acids (e.g.,genes, gene fragments, gene regulatory sequences, and antisensemolecules), nucleoproteins, polysaccharides, glycoproteins, andlipoproteins. Classes of biologically active compounds that can beincorporated into the article of manufacture described herein include,without limitation, anticancer agents, antibiotics, analgesics,anti-inflammatory agents, immunosuppressants, enzyme inhibitors,antihistamines, anti-convulsants, hormones, muscle relaxants,antispasmodics, ophthalmic agents, prostaglandins, anti-depressants,anti-psychotic substances, trophic factors, osteoinductive proteins,growth factors, and vaccines.

In some cases the article of manufacture, e.g. an orthopedic implantneeds to be tuned to degrade in a shorter time frame to allow fordynamic transfer of the load back to the healing bone. This could beaccomplished numerous different ways such as autoclaving multiple timesto degrade the silk fibroin or incorporating enzymes into the constructsthat activate upon hydration [28, 33, 34]. Coating the silk devices withbioactive compounds such as BMP-2 nd other pharmaceuticals could providebenefits in bone fixation systems. The article of manufacture can alsoincorporate bioactive compounds such as BMP-2 or antibiotics to promotebone ingrowth [29-31]. Without wishing to be bound by a theory, it isbelieved that such factors can be used to modulate healing and promoteremodeling of bone.

The combination of the silk with bioactive compounds such as enzymes,bone morphogenctic protein 2 (BMP-2), and pharmaceuticals is believed toprovide multifunctional benefits not currently utilized in bone fixationsystems.

Generally, any therapeutic agent can be encapsulated in the drugdelivery vehicle or composition comprising the article of manufacturedescribed herein. As used herein, the term “therapeutic agent” means amolecule, group of molecules, complex or substance administered to anorganism for diagnostic, therapeutic, preventative medical, orveterinary purposes. As used herein, the term “therapeutic agent”includes a “drug” or a “vaccine.” This term include externally andinternally administered topical, localized and systemic human and animalpharmaceuticals, treatments, remedies, nutraceuticals, cosmeceuticals,biologicals, devices, diagnostics and contraceptives, includingpreparations useful in clinical and veterinary screening, prevention,prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy,surgery, monitoring, cosmetics, prosthetics, forensics and the like.This term can also be used in reference to agriceutical, workplace,military, industrial and environmental therapeutics or remediescomprising selected molecules or selected nucleic acid sequences capableof recognizing cellular receptors, membrane receptors, hormonereceptors, therapeutic receptors, microbes, viruses or selected targetscomprising or capable of contacting plants, animals and/or humans. Thisterm can also specifically include nucleic acids and compoundscomprising nucleic acids that produce a therapeutic effect, for exampledeoxyribonucleic acid (DNA), ribonucleic acid (RNA), or mixtures orcombinations thereof, including, for example, DNA nanoplexes, siRNA,shRNA, aptamers, ribozymes, decoy nucleic acids, antisense nucleicacids, RNA activators, and the like.

The term “therapeutic agent” also includes an agent that is capable ofproviding a local or systemic biological, physiological, or therapeuticeffect in the biological system to which it is applied. For example, thetherapeutic agent can act to control infection or inflammation, enhancecell growth and tissue regeneration, control tumor growth, act as ananalgesic, promote anti-cell attachment, and enhance bone growth, amongother functions. Other suitable therapeutic agents can includeanti-viral agents, hormones, antibodies, or therapeutic proteins. Othertherapeutic agents include prodrugs, which are agents that are notbiologically active when administered but, upon administration to asubject are converted to biologically active agents through metabolismor some other mechanism. Additionally, a silk-based drug deliverycomposition can contain combinations of two or more therapeutic agents.

A therapeutic agent can include a wide variety of different compounds,including chemical compounds and mixtures of chemical compounds, e.g.,small organic or inorganic molecules; saccharines; oligosaccharides;polysaccharides; biological macromolecules, e.g., peptides, proteins,and peptide analogs and derivatives; peptidomimetics; antibodies andantigen binding fragments thereof; nucleic acids; nucleic acid analogsand derivatives; an extract made from biological materials such asbacteria, plants, fungi, or animal cells; animal tissues; naturallyoccurring or synthetic compositions; and any combinations thereof, insome embodiments, the therapeutic agent is a small molecule.

As used herein, the term “small molecule” can refer to compounds thatare “natural product-like,” however, the term “small molecule” is notlimited to “natural product-like” compounds. Rather, a small molecule istypically characterized in that it contains several carbon-carbon bonds,and has a molecular weight of less than 5000 Daltons (5 kDa), preferablyless than 3 kDa, still more preferably less than 2 kDa, and mostpreferably less than 1 kDa. In some cases it is preferred that a smallmolecule have a molecular weight equal to or less than 700 Daltons.

Exemplary therapeutic agents include, but are not limited to, thosefound in Harrison's Principles of Internal Medicine, 13^(th) Edition,Eds. T. R. Harrison et al. McGraw-Hill N.Y., N.Y., Physicians' DeskReference, 50^(th) Edition, 1997, Oradell N.J., Medical Economics Co.;Pharmacological Basis of Therapeutics, 8^(th) Edition, Goodman andGilman, 1990; United States Pharmacopeia, The National Formulary, USPXII NF XVII, 1990, the complete contents of all of which areincorporated herein by reference.

Therapeutic agents include the herein disclosed categories and specificexamples. It is not intended that the category be limited by thespecific examples. Those of ordinary skill in the art will recognizealso numerous other compounds that fall within the categories and thatare useful according to the present disclosure. Examples include aradiosensitizer, a steroid, xanthine, a beta-2-agonist bronchodilator,an anti-inflammatory agent, an analgesic agent, a calcium antagonist, anangiotensin-converting enzyme inhibitors, a beta-blocker, a centrallyactive alpha-agonist, an alpha-1-antagonist, ananticholinergic/antispasmodic agent, a vasopressin analogue, anantiarrhythmic agent, an antiparkinsonian agent, anantiangina/antihypertensive agent, an anticoagulant agent, anantiplatelet agent, a sedative, an ansiolytic agent, a peptidic agent, abiopolymeric agent, an antineoplastic agent, a laxative, anantidiarrheal agent, an antimicrobial agent, an antifungal agent, avaccine, a protein, or a nucleic acid. In a further aspect, thepharmaceutically active agent can be coumarin, albumin, steroids such asbetamethasone, dexamethasone, methylprednisolone, prednisolone,prednisone, triamcinolone, budesonide, hydrocortisone, andpharmaceutically acceptable hydrocortisone derivatives; xanthines suchas theophylline and doxophylline; beta-2-agonist bronchodilators such assalbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol;antiinflammatory agents, including antiasthmatic anti-inflammatoryagents, antiarthritis antiinflammatory agents, and non-steroidalantiinflammatory agents, examples of which include but are not limitedto sulfides, mesalamine, budesonide, salazopyrin, diclofenac,pharmaceutically acceptable diclofenac salts, nimesulide, naproxene,acetaminophen, ibuprofen, ketoprofen and piroxicam; analgesic agentssuch as salicylates; calcium channel blockers such as nifedipine,amlodipine, and nicardipine; angiotensin-converting enzyme inhibitorssuch as captopril, benazepril hydrochloride, fosinopril sodium,trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride,and moexipril hydrochloride; beta-blockers (i.e., beta adrenergicblocking agents) such as sotalol hydrochloride, timolol maleate, esmololhydrochloride, carteolol, propanolol hydrochloride, betaxololhydrochloride, penbutolol sulfate, metoprolol tartrate, metoprololsuccinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprololfumarate; centrally active alpha-2-agonists such as clonidine;alpha-1-antagonists such as doxazosin and prazosin;anticholinergic/antispasmodic agents such as dicyclomine hydrochloride,scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate,and oxybutynin; vasopressin analogues such as vasopressin anddesmopressin; antiarrhythmic agents such as quinidine, lidocaine,tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamilhydrochloride, propafenone hydrochloride, flecainide acetate,procainamide hydrochloride, moricizine hydrochloride, and disopyramidephosphate; antiparkinsonian agents, such as dopamine, L-Dopa/Carbidopa,selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine, andbromocryptine; antiangina agents and antihypertensive agents such asisosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol andverapamil; anticoagulant and antiplatelet agents such as Coumadin,warfarin, acetylsalicylic acid, and ticlopidine; sedatives such asbenzodiazapines and barbiturates; ansiolytic agents such as lorazepam,bromazepam, and diazepam; peptidic and biopolymeric agents such ascalcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin,insulin, somatostatin, protirelin, interferon, desmopressin,somatotropin, thymopentin, pidotimod, erythropoietin, interleukins,melatonin, granulocyte/macrophage-CSF, and heparin; antineoplasticagents such as etoposide, etoposide phosphate, cyclophosphamide,methotrexate, 5-fluorouracil, vincristine, doxorubicin, cisplatin,hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase,altretamine, mitotane, and procarbazine hydrochloride; laxatives such assenna concentrate, casanthranol, bisacodyl, and sodium picosulphate;antidiarrheal agents such as difenoxine hydrochloride, loperamidehydrochloride, furazolidone, diphenoxylate hydrochloride, andmicroorganisms; vaccines such as bacterial and viral vaccines;antimicrobial agents such as penicillins, cephalosporins, andmacrolides, antifungal agents such as imidazolic and triazolicderivatives; and nucleic acids such as DNA sequences encoding forbiological proteins, and antisense oligonucleotides.

Anti-cancer agents include alkylating agents, platinum agents,antimetabolites, topoisomerase inhibitors, antitumor antibiotics,antimitotic agents, aromatase inhibitors, thymidylate synthaseinhibitors, DNA antagonists, farnesyltransferase inhibitors, pumpinhibitors, histone acetyltransferase inhibitors, metalloproteinaseinhibitors, ribonucleoside reductase inhibitors, TNP alphaagonists/antagonists, endothelinA receptor antagonists, retinoic acidreceptor agonists, immuno-modulators, hormonal and antihormonal agents,photodynamic agents, and tyrosine kinase inhibitors.

Antibiotics include aminoglycosides (e.g., gentamicin, tobramycin,netilmicin, streptomycin, amikacin, neomycin), bacitracin, corbapenems(e.g., imipenem/cislastatin), cephalosporins, colistin, methenamine,monobactams (e.g., aztreonam), penicillins (e.g., penicillin G,penicillin V, methicillin, natcillin, oxacillin, cloxacillin,dicloxacillon, ampicillin, amoxicillin, carbenicllin, ticarcillin,piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, andvancomycin; and bacteriostatic agents such as chloramphenicol,clindanyan, macrolides (e.g., erythromycin, azithromycin,clarithromycin), lincomyan, nitrofurantoin, sulfonamides, tetracyclines(e.g., tetracycline, doxycycline, minocycline, demeclocyline), andtrimethoprim. Also included are metronidazole, fluoroquinolones, andritampin.

Enzyme inhibitors are substances which inhibit an enzymatic reaction.Examples of enzyme inhibitors include edrophonium chloride.N-methylphysostigmine, neostigmine bromide, physostigmine sulfate,tacrine, tacrine, 1-hydroxy maleate, iodotubercidin,p-bromotetramiisole, 10-(alpha-diethylaminopropionyl)-phenothiaxinehydrochloride, calmidazolium chloride,hemicholinium-3,3,5-dinitrocatechol, diacylglycerol kinase inhibitor I,diacylglycerol kinase inhibitor II, 3-phenylpropargylmaine,N°-monomethyl-Larginine acetate, carbidopa, 3-hydroxybenzylhydrazine,hydralazine, clorgyline, deprenyl, hydroxylamine, iproniazid phosphate,6-MeO-tetrahydro-9H-pyrido-indol, nialamide, parglyline, quinacrine,semicarbazide, tranylcypromise,N,N-diethyalminoethyl-2,2-diphenylvalerate hydrochloride,3-isobutyl-1-methylxanthne, papaverine, indomethacind,2-cyclooctyl-2-hydroxyethylamine hydrochloride,2,3-dichloro-a-methylbenzylamine (DCMB),8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride; p-aminoglutethimide, p-aminoglutethimide tartrate, 3-iodotyrosine,alpha-methyltyrosine, acetazolamide, dichlorphenamide,6-hydroxy-2-benzothiazolesulfonamide, and allopurinol.

Antihistamines include pyrilamine, chlorpheniramine, andtetrahydrazoline, among others.

Anti-inflammatory agents include corticosteroids, nonsteroidalanti-inflammatory drugs (e.g., aspirin, phenylbutazone, indomethacin,sulindac, tolmetin, ibuprofen, piroxicam, and fenamates), acetaminophen,phenacetin, gold salts, chloroquine, D-Penicillamine, methotrexatecolchicine, allopurinol, probenecid, and sulfinpyrazone.

Muscle relaxants include mephenesin, methocarbomal, cyclobenzaprinehydrochloride, trihexylphenidyl hydrochloride, levodopa/carbidopa, andbiperiden.

Anti-spasmodics include atropine, scopolamine, oxyphenonium, andpapaverine.

Analgesics include aspirin, phenylbutazone, idomethacin, sulindac,tolmetic, ibuprofen, piroxicam, fenamates, acetaminophen, phenacetin,morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids(e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate,loperamide, morphine sulfate, noscapine, norcodeine, normorphine,thebaine, norbinaltorphimine, buprenorphine, chlomaltrexamine,funaltrexamione, nalbuphine, nalorphine, naloxone, naloxonazine,naltrexone, and naltrindole), procaine, lidocain, tetracaine anddibucaine.

Ophthalmic agents include sodium fluorescein, rose bengal, methacholine,adrenaline, cocaine, atropine, alpha-chymotrypsin, hyaluronidase,betaxalol, pilocarpine, timolol, timolol salts, and combinations thereof

Prostaglandins are art recongized and are a class of naturally occurringchemically related, long-chain hydroxy fatty acids that have a varietyof biological effects.

Anti-depressants are substances capable of preventing or relievingdepression. Examples of anti-depressants include imipramine,amitriplyline, nortriptyline, protriptyline, desipramine, amoxapine,doxepin, maprotiline, tranyleypromine, phenelzine, and isocarboxazide.

Trophic factors are factors whose continued presence improves theviability or longevity of a cell. Trophic factors include, Withoutlimitation, platelet-derived growth factor (PDGP), neutrophil-activatingprotein, monocyte chemoattractant protein, macrophage-inflammatoryprotein, platelet factor, platelet basic protein, and melanoma growthstimulating activity; epidermal growth factor, transforming growthfactor (alpha), fibroblast growth factor, platelet-derived endothelialcell growth factor, insulin-like growth factor, glial derived growthneurotrophic factor, ciliary neurotrophic factor, nerve growth factor,bone growth/cartilage-inducing factor (alpha and beta), bonemorphogenetic proteins, interleukins (e.g., interleukine inhibitors orinterleukine receptors, including interleukin 1 through interleukin 10),interferons (e.g., interferon alpha, beta and gamma), hematopoieticfactors, including erythropoietin, granulocyte colony stimulatingfactor, macrophage colony stimulating factor and granulocyte-macrophagecolony stimulating factor; tumor necrosis factors, and transforminggrowth factors (beta), including beta-1, beta-2, beta-3, and activin.

Hormones include estrogens (e.g, estradiol, estrone, estriol,diethylstibestrol, quinestrol, chlorotrianisene, ethinyl estradiol,mestranol), anti-estrogens (e.g., clomiphene, tamoxifen), progestins(e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone,norgestrel), antiprogestin (mifepristone), androgens (e.g. testosteronecypionate, fluoxymesterone, danazol, testolactone), anti-androgens(e.g., cyproterone acetate, flutamide), thyroid hormones (e.g,triodothyronne, thyroxine, propylthiouracil, methimazole, and iodixode),and pituary pituitary hormones (e.g., corticotropin, sumutotropin,oxytocin, and and vasopressin). Hormones are commonly employed inhormone replacement therapy and or for purposes of birth control.Steroid hormones, such as prednisone, are also used asimmunosuppressants and anti-inflammatories.

The biologically active can be an osteogenic protein. Accordingly, insome embodiments, the biologically active agent is desirably selectedfrom the family of proteins known as the transforming growth factorsbeta (TGF-[3) superfamily of proteins, which includes the activins,inhibins and bone morphogenetic proteins (BMPs). Most preferably, theactive agent includes at least one protein selected from the subclass ofproteins known generally as BMPs, which have been disclosed to haveosteogenic activity, and other growth and differentiation typeactivities. These BMPs include BMP proteins BMP-2, BMP-3, BMP-4, BMP5,BMP-6 and BMP-7, disclosed for instance in U.S. Pat. Nos. 5,108,922;5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8,disclosed in PCT publication WO91/18098; and BMP-9, disclosed in PCTpublication WO93/00432, BMP-10, disclosed in PCT application WO94/26893;BMP-11, disclosed in PCT application WO94/26892, or BMP-12 or BMP-13,disclosed in PCT application WO 95/16035; BMP-14; BMP-15, disclosed inU.S. Pat. No. 5,635,372; or BMP-16, disclosed in U.S. Pat. No.5,965,403. Other TGF-β proteins, which can be used include Vgr-2, Joneset al., Mol. Endocrinol. 611961 (1992), and any of the growth anddifferentiation factors (GDFs), including those described in PCTapplications WO94/15965; WO94/15949; WO95/01801; WO95/01802; WO94/21681;WO94/15966; WO95/10539; WO96/01845; WO96/02559 and others. Also usefulin the invention can be BIP, disclosed in WO94/01557; HP00269, disclosedin JP Publication number: 7-250688; and BMP-14 (also known as MP52,CDMP1, and GDF5), disclosed in PCT application WO93/16099. Thedisclosures of all of the above applications are incorporated herein byreference. Subsets of BMPs which can be used include BMP-2, BMP-3,BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11,BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, BMP-17, and BMP18. Otherosteogenic agents known in the art can also be used, such asteriparatide (FORTEO™), CHRYSALIN®, prostaglandin E2, or LIM protein,among others.

The biologically active agent can be recombinantly produced, or purifiedfrom a protein composition. The active agent, if a TGF-β such as a BMP,or other dimeric protein, can be homodimeric, or can be heterodimericwith other BMPs (e.g., a heterodimer composed of one monomer each ofBMP-2 and BMP-6) or With other members of the TGF-β superfamily, such asactivins, inhibins, and TGF-β1 (e.g., a heterodimer composed of onemonomer each of a BMP and a related member of the TGF-β superfamily).Examples of such heterodimeric proteins are described for example inPublished PCT Patent Application WO 93/09229, the content of which isincorporated herein by reference.

The active agent can further include additional agents such as theHedgehog, Frazzled, Chordin, Noggin, Cerberus and Follislatin proteins.These families of proteins are generally described in Sasai et al., Cell791779-790 (1994) (Chordin); PCT Patent Publication WO94/05800 (Noggin);and Fukui et al., Devel. Biol. 159: 1 31 (1993) (Follistatin). Hedgehogproteins are described in WO96/16668; WO96/17924; and WO95/18856. TheFrazzled family of proteins is a recently discovered family of proteinsWith high homology to the extracellular binding domain of the receptorprotein family known as Frizzled. The Frizzled family of genes andproteins is described in Wang et al., J. Biol. Chem. 271 :44684476(1996). The active agent can also include other soluble receptors, suchas the truncated soluble receptors disclosed in PCT patent publicationWO95/07982. From the teaching of WO95/07982, one skilled in the art willrecognize that truncated soluble receptors can be prepared for numerousother receptor proteins. The above publications are hereby incorporatedby reference herein.

The amount of osteogenic protein effective to stimulate increasedosteogenic activity of present or infiltrating progenitor or other cellswill depend upon the size and nature of the defect being treated, aswell as the carrier being employed. Generally, the amount of protein tobe delivered is in a range of from about 0.1 to about 100 mg; preferablyabout 1 to about 100 mg; most preferably about 10 to about 80 mg.

Biologically active agents can be introduced into the article ofmanufacture described herein during or after its formation. Agents canconveniently be mixed into the starting solution prior to fabrication ofthe article of manufacture described herein. Alternatively, the articleof manufacture described herein can be fabricated, shaped into a desiredshape, and then exposed to the biologically active agent in solution.This particular approach is particularly well suited for proteins, whichare known to have an affinity for apatitic materials. A buffer solutioncontaining the biologically active agent can be employed, instead ofwater, as the aqueous solution in which the article of manufacturedescribed herein is, or example, irrigated prior to use. Buffers can beused in any pH range, but most often will be used in the range of 5.0 to8.0 in preferred embodiments the pH will be compatible with prolongedstability and efficacy of the desired biologically active agent and, inmost preferred embodiments, will be in the range of 5.5 to 7.4. Suitablebuffers include, but are not limited to, carbonates, phosphates (e.g.,phosphate buffered saline), and organic buffers such as Tris, HEPES, andMOPS. Most often, the buffer will be selected for its biocompatibililywith the host tissues and its compatibility with the biologically activeagent. For most applications of nucleic acids, peptides or antibiotics asimple phosphate buffered saline can suffice.

Standard protocols and regimens for delivery of the above listed agentsare known in the art. Typically, these protocols are based on oral orintravenous delivery. Biologically active agents are introduced into thevehicle in amounts that allow delivery of an appropriate dosage of theagent to the implant site. In most cases, dosages are determined usingguidelines known to practitioners and applicable to the particular agentin question. The exemplary amount of biologically active agent to beincluded in the the article of manufacture described herein is likely todepend on such variables as the type and extent of the condition, theoverall health status of the particular patient, the formulation of theactive agent, and the bioresorbability of the delivery vehicle used.Standard clinical trials may be used to optimize the dose and dosingfrequency for any particular biologically active agent

Generally, any amount of the supplementary material, such as abiocompatible polymer, biologically active agent, and therapeutic agentcan be loaded into the article of manufacture described herein, forexample, from about 0.1 ng to about 1000 mg of the therapeutic agent canbe loaded in the article of manufacture described herein. In someembodiment, amount of the supplementary in the silk solution, silkcomposition or the article of manufacture is selected from the rangeabout from 0.001% (w/w) up to 95% (w/w), preferably, from about 5% (w/w)to about 75% (w/w), and most preferably from about 10% (w/w) to about60% (w/w) of the total composition. In some embodiments, amount ofamount of the supplementary in the article of manufacture describedherein is from about 0.01% to about 95% (w/v), from about 0.1% to about90% (w/w), from about 1% to about 85% (w/w), from about 5% to about 75%(w/w), from about 10% to about 65% (w/w), or from about 10% to about 50%(w/w), of the total composition.

In some embodiments, amount of the supplementary in the article ofmanufacture described herein is from about 1% to about 99% (w/w), fromabout 0.05% to about 99% (w/w), from about 0.1% to about 90% (w/w), fromabout 0.5% to about 85% (w/w), from about 5% to about 80% (w/w), fromabout 10% to about 60% (w/w) of the total composition. In someembodiments, amount of the supplementary in the silk solution, the silkcomposition or the article of manufacture is from about 0.1% to about99% (w/w), from about 1% to about 90% (w/w), from about 2% to about 80%(w/w), from about 5% to about 75% (w/w), from about 5% to about 50%(w/w), from about 0.055% to about 0.1% (w/w) of the total composition.

After preparation, the article of manufacture described herein can besterilized using conventional sterilization process such asradiation-based sterilization (i.e. gamma-ray), chemical basedsterilization (ethylene oxide), autoclaving, or other appropriateprocedures. In some embodiments, sterilization process can be withethylene oxide at a temperature between from about 52° C. to about 55°C. for a time of 8 or less hours. The the article of manufacturedescribed herein can also be processed aseptically. Sterile article ofmanufacture described herein can be packaged in an appropriate sterilizemoisture resistant package for shipment.

Without wishing to be bound by a theory, the article of manufacturedescribed herein provides a number of advantages. The material canwithstand physiological loading forces; can initiate new bone formationand stimulate healing through direct bone-silk interface; can promoteosteogenesis by local delivery of bone morphogenic growth factors; andcan achieve complete graft resorption and non-union closure. The methodsand articles of manufacture prepared using the same provide a numberadvantages over the prior art.

Embodiments of the invention can be described by any of the followingparagraphs:

-   -   1. A method comprising:        -   (i) providing a composition comprising silk particles; and    -   (ii) compacting the composition by application of pressure into        a solid state.    -   2. The method of paragraph 1, wherein the silk particles are        nanoparticles or microparticles.    -   3. The method of paragraph 1 or 2, wherein the composition        comprises silk in an amount of about 25% (w/w) or higher.    -   4. The method of any of paragraphs 1-3, wherein said pressure is        at least 0.05 bar.    -   5. The method of any of paragraphs 1-4, wherein said compacting        is at an elevated temperature.    -   6. The method of paragraph 5, wherein the elevated temperature        is at least 30° C.    -   7. The method of any of paragraphs 1-6, wherein the composition        further comprises a binder.    -   8. The method of paragraph 7, wherein the binder is a liquid.    -   9. The method of paragraph 7 or 8, wherein the binder is water.    -   10. The method of any of paragraphs 7-9, wherein the composition        comprises from about 0.1% (w/vv) to about 50% (w/w) of the        binder.    -   11. The method of any of paragraphs 1-10, wherein the silk        particles comprise degummed silk.    -   12. The method of any of paragraphs 1-11, wherein the silk        particles comprise non-degummed silk.    -   13. The method of any of paragraphs 1-12, wherein the        composition comprises a mixture of silk particles comprising        degummed silk and silk particles comprising non-degummed silk.    -   14. The method of paragraph 13, wherein ratio of dcgumnicd silk        to non-degummed silk is from about 50:1 to about 1:50 (w/w).    -   15. The method of paragraph 13 or 14, wherein the ratio of        degummed silk to non-degummed silk is from about 1:1 to about        1:20.    -   16. The method of any of paragraphs 1-15, wherein the        composition further comprises an additive.    -   17. The method of paragraph 16, wherein the additive is selected        from the group consisting of small organic or inorganic        molecules; saccharines; oligosaccharides; polysaccharides;        biological macromolecules, e.g., peptides, proteins, and peptide        analogs and derivatives; peptidomimetics; antibodies and antigen        binding fragments thereof; nucleic acids; nucleic acid analogs        and derivatives; glycogens or other sugars; immunogens;        antigens; an extract made from biological materials such as        bacteria, plants, fungi, or animal cells; animal tissues;        naturally occurring or synthetic compositions; and any        combinations thereof.    -   18. The method of paragraph 16 or 17, wherein the additive is in        a form selected from the group consisting of a particle, a        fiber, a film, a gel, a hydrogel, a mesh, a mat, a non-woven        mat, a powder, a fabric, a scaffold, a tube, a slab or block, a        fiber, a foam or a sponge, a needle, a lyophilized article, and        any combinations thereof.    -   19. The method of paragraph 18, wherein the additive particle is        a nanoparticle or a microparticle.    -   20. The method of any of paragraphs 16-19, wherein the additive        is a silk-based material.    -   21. The method of paragraph 20, wherein the silk-based material        is selected from the group consisting of silk particles, silk        fibers, micro-sized silk fibers, unprocessed silk fibers, and        any combinations thereof.    -   22. The method of any of paragraphs 16-21, wherein the        composition comprises from about 0.1% to (w/w) to about 99%        (w/w) of the additive.    -   23. The method of any of paragraphs 16-22, wherein ratio of silk        to the additive is from about 10:1 to about 1:10 (w/w).    -   24. The method of any of paragraphs 16-23, wherein the additive        is an active agent.    -   25. The method of paragraph 24, wherein the active agent is a        therapeutic agent.    -   26. The method of any of paragraphs 1-25, wherein the        composition is in a mold.    -   27. The method of any of paragraphs 1-26, further comprising        processing the composition to a desired shape after said        compacting step.    -   28. The method paragraph 27, wherein said processing is        machining, turning (lathe), rolling, thread rolling, drilling,        milling, sanding, punching, die cutting, blanking, broaching,        and any combinations thereof.    -   29. The method of any of paragraphs 1-28, further comprising        inducing a conformational change in silk fibroin to a beta-sheet        conformation.    -   30. The method of paragraph 29, wherein said inducing a        conformational change comprises solvent immersion, water        annealing, water vapor annealing, sonication, pH reduction,        exposure to an electric field, controlled slow drying,        freeze-drying, compressing, heating, application of shear        stress, and any combinations thereof.    -   31. An article of manufacture comprising silk obtained by the        method of any of paragraphs 1-30.    -   32. The article of manufacture of paragraph 31, wherein the        article of manufacture is a medical device.    -   33. The article of manufacture of paragraph 32, wherein the        medical device is selected from the group consisting of an        orthopedic implant, a facial implant, a nasal implant, a suture        anchor, a dental implant, a Swanson prosthetic, and any        combinations thereof.    -   34. The article of manufacture of paragraph 33, wherein said        orthopedic implant is selected from the group consisting of an        orthopedic screw, an orthopedic plate, an orthopedic rod, an        orthopedic tulip, and any combinations thereof.    -   35. The article of manufacture of any of paragraphs 31-34,        wherein the article of manufacture is a tapping screw.    -   36. The article of manufacture of any of paragraphs 31-35,        wherein the article of manufacture is osteocondcutive,        osteoinductive, osteogenic, or any combinations thereof.    -   37. The article of manufacture of any of paragraphs 31-36,        wherein the article of manufacture is bioresorbable.    -   38. An article of manufacture comprising silk, wherein the        article has a compressive strength of at least 5 MPa; a        compressive elastic modulus (Young's modulus) of at least 100        MPa; a shear strength of at least 104 MPa; or a density of at        least 1.1 g/cm³.    -   39. The article of manufacture of paragraph 38, wherein the        article of manufacture is obtained by a method of any of        paragraphs 1-30.    -   40. A method for increasing compressive strength, elastic        modulus, flexural stiffness, or shear stiffness of a silk-based        material, the method comprising:        -   (i) providing a composition comprising silk particles; and        -   (ii) compacting the composition by application of pressure            into a solid state.

SOME SELECTED DEFINITIONS

For convenience, certain icons employed herein, in the specification,examples and appended claims are collected herein. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the an to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

The term “herein” is meant to include all of the disclosure and is notintended to be limited to a subsection of the disclosure.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean ±5% of the value being referred to. For example, about 100 meansfrom 95 to 105.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means at least two standarddeviation (2SD) away from a reference level. The term refers tostatistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true.

The term “blank” as used herein means an unfinished part of simplegeometry that can later be modified by various machining methods tocreate the desired shape of the article of manufacture.

The term “drying” means removal of at least a portion of any liquidcarrier.

The term “bone repair” refers to any procedure for repairing bone,including those which use a material as a substitute for bone grafts.

The term “bone augmentation” refers to the use of any procedure foradding or building bone.

The term “bone replacement” refers to the use of any procedure forreplacing existing bone.

As used herein, the term “microparticle” refers to a particle having aparticle size of about 0.01 μm to about 1000 μm.

As used herein, the term “nanoparticle” refers to particle having aparticle size of about 0.1 nm to about 1000 nm.

It will be understood by one of ordinary skill in the art that particlesusually exhibit a distribution of particle sizes around the indicated“size.” Unless otherwise stated, the term “particle size” as used hereinrefers to the mode of a size distribution of particles, i.e., the valuethat occurs most frequently in the size distribution. Methods formeasuring the particle size are known to a skilled artisan, e.g., bydynamic light scattering (such as photocorrelation spectroscopy, laserdiffraction, low-angle laser light scattering (LALLS), and medium-anglelaser light scattering (MALLS)), light obscuration methods (such asCoulter analysis method), or other techniques (such as theology, andlight or electron microscopy).

In some embodiments, the particles can be substantially spherical. Whatis meant by “substantially spherical” is that the ratio of the lengthsof the longest to the shortest perpendicular axes of the particle crosssection is less than or equal to about 1.5. Substantially spherical doesnot require a line of symmetry. Further, the particles can have surfacetexturing, such as lines or indentations or protuberances that are smallin scale when compared to the overall size of the particle and still besubstantially spherical. In some embodiments, the ratio of lengthsbetween the longest and shortest axes of the particle is less than ofequal to about 1.5, less than or equal to about 1.45, less than or equalto about 1.4, less than or equal to about 1.35, less than or equal toabout 1.30, less than or equal to about 1.25, less than or equal toabout 1.20, less than or equal to about 1.15 less than or equal to about1.1. Without wishing to be bound by a theory, surface contact isminimized in particles that are substantially spherical, which minimizesthe undesirable agglomeration of the particles upon storage. Manycrystals or flakes have flat surfaces that can allow large surfacecontact areas where agglomeration can occur by ionic or non-ionicinteractions. A sphere permits contact over a much smaller area.

In some embodiments, the particles have substantially the same particlesize. Particles having a broad size distribution where there are bothrelatively big and small particles allow for the smaller particles tofill in the gaps between the larger particles, thereby creating newcontact surfaces. A broad size distribution can result in larger spheresby creating many contact opportunities for binding agglomeration. Theparticles described herein are within a narrow size distribution,thereby minimizing opportunities for contact agglomeration. What ismeant by a “narrow size distribution” is a particle size distributionthat has a ratio of the volume diameter of the 90th percentile of thesmall spherical particles to the volume diameter of the 10th percentileless than or equal to 5. In some embodiments, the volume diameter of the90th percentile of the small spherical particles to the volume diameterof the 10th percentile is less than or equal 4.5, less than or equal to4, less than or equal to 3.5, less than or equal to 3, less than orequal to 2.5, less than or equal to 2, less than or equal to 1.5, lessthan or equal to 1.45, less than or equal to 1.40, less than or equal to1.35, less than or equal to 1.3, less than or equal to 1.25, less thanor equal to 1.20, less than or equal to 1.15, or less than or equal to1.1.

Geometric Standard Deviation (GSD) can also be used to indicate thenarrow size distribution. GSD calculations involved determining theeffective cutoff diameter (ECD) at the cumulative less than percentagesof 15.9% and 84.1%. GSD is equal to the square root of the ratio of theECD less than 84.17% to ECD less than 15.9%. The GSD has a narrow sizedistribution when GSD<2.5. In some embodiments, GSD is less than 2, lessthan 1.75, or less than 1.5. In one embodiment, GSD is less than 1.8.

The disclosure is further illustrated by the following examples, whichshould not be construed as limiting. The examples are illustrative only,and are not intended to limit, in any manner, any of the aspectsdescribed herein. The following examples do not in any way limit theinvention.

EXAMPLES Example 1. Exemplary Methods Used for Making Compacted SilkArticles

Fabrication of aluminum powder compaction press and first sample. Thecompaction press was designed in Solidworks and fabricated from aluminumalloys, except for the piston, which was made from steel. The entireassembly is held together with ¼″-20 screws, which also are used toapply requisite pressure to the piston/sample. The initial sample wasmade using silk powder that had been pulverized and ball milled. A totalof 25% of the powder was generated from degummed silk fibroin, while theremaining 75% was from a non-degumming fibroin source. A total of about4 grams of powder was mixed with 2 ml of distilled water. The assembledcompaction press was stoted in an oven for 48 hours at 60° C. The firstsample had an excellent disk shape, with smooth surfaces, a dull surfacefinish, and relatively light coloration. Upon cooling, the sample couldnot be broken by hand, indicating excellent toughness and strengthproperties.

Fabrication of an acrylic compaction press. In order to improve theprocess and make fabrication easier, a new compaction press was designedto be fabricated out of acrylic. In addition, the acrylic material couldbe cut on a Trotec Speedy 300 laser etcher, allowing for rapidreproduction of future press components. Given the acrylic was not heatresistance, only room temperature testing was appropriate.

Sample fabrication I using acrylic compaction press. A silk powdermixture consisting of 10% degummed fibroin and 90% non-degummed fibroinwas utilized. Approximately 5 grams of powder was mixed with 2.5 ml ofdistilled water and silk solution. The press was then clamped lightly ina vise for 3 days at ambient temperature. When released from the press,the silk construct was not completely dry, and therefore it cracked andflaked. Material that had not flaked away was relatively stiff, butextremely brittle (and easily crumbled under pressure).

Sample fabrication II using acrylic compaction press. A second samplewas created using the acrylic press. Instead of ball milled powder,however, commercial pure silk powder was used. This powder wasfabricated using hydrolysis, not milling. This powder was very easy tosolubulize in distilled water and had a much whiter color than themilled powder. As in the prior experiment, silk powder made of 10%degummed silk fibroin and 90% non-degummed fibroin was utilized.Combining approximately 5 grams of powder with 2.5 ml distilled water,the mixture was clamped in the acrylic press for 4 days. The final silkconstruct was whiter than the construct fabricated above in Samplefabrication I using acrylic compaction press and the mechanicalproperties seemed to be worse, with the construct having a chalky feeland the characteristic of crumbling very easily.

Sample at higher temperature using aluminum compaction press. Using thealuminum compaction press, and a powder mixture of 10% degummed silkfibroin/90% non-degummed fibroin, approximately 3 grams of powder weremixed with 2 ml of distilled water. The sample/press was stored in anoven at 120° C. for 48 hours. The resulting sample was darker andappeared to be stiffer and somewhat translucent. The hardnessqualitatively seemed higher than with previous samples. The darker colorcan be attributed to the much higher heat that potentially caused someburning of silk fibroin.

Compaction sample I using water and silk solution. Using the aluminumcompaction press and a powder mixture of 25% degummed silk fibroin and75% non-degummed silk fibroin, approximately 3 grams of powder was mixedwith 2.5 ml of distilled water and silk solution. Given the silksolution concentration was approximately 7% w/v, the powder wasessentially mixed with a 3.5% w/v silk solution (the distilled wateracts to dilute the silk fibroin concentration). The sample and presswere stored in an oven at 120° C. for 48 hours. The resulting constructwas somewhat burned and cracked. The darker coloration is attributed tothe higher temperature (earlier samples using no heating evidenced nocolor change). Without wishing to be bound by theory. The crack may bedeveloped as a result of the addition of silk solution.

Compaction sample II using water and silk solution. A silk sample wasproduced using the aluminum compaction press, a powder mixture of 25%degummed silk fibroin and 75% non-degummed silk fibroin, and a dilutesilk solution (distilled water added to silk solution). Approximately 3grams of powder was combined wilh a total of 2.5 ml dilute silksolution. After storing in an oven at 120° C. oven for 48 hours, theresulting construct was removed. The construct had a glassy surface, wassomewhat translucent, and contained many serious cracks. It is likelythat the glass-like appearance is due to the temperatures exceeding thematerial's glass transition temperature. Without wishing to be bound bytheory, the cracking can be linked to the addition of silk solutioninstead of pure water as a binding agent.

New test protocol to develop silk powder compaction. To betterunderstand the effect of various processing parameters on the propertiesof the resulting silk samples, a test protocol was developed. The twoparameters investigated are the specific powder blends of degummed andnon-degummed silk fibroin, as well as the amount of distilled water used(no silk solution was to be added in this series of experiments). Thefirst sample created with the test protocol utilized a powder blend of10% degummed and 90% non-degummed silk fibroin. The powder was mixedwith 1 ml of distilled water and the sample/press was stored in on ovenat 60° C. oven for 24 hours. One side of the resulting construct wasblack while slightly translucent and lighter in color on the other. Thediscoloration was attributed to corrosion that had occurred on the steelpiston. The sample was observed to be fairly homogeneous, exhibited highstrength and did not contain cracks or flakes.

The second sample created with the test protocol utilized a powder blendof 15% degummed and 85% non-degummed silk fibroin was utilized.Approximately 3 grams of powder was mixed with 2 ml of distilled waterand the sample/press was stored in an oven at 90° C. oven for 24 hours.The time in the oven was insufficient to allow the sample to completelydry. It warped and flaked once it started drying outside of the press inambient conditions. Once dry, the remaining construct exhibited goodproperties (could not be fractured using hand pressure).

The third sample created with the test protocol utilized a powder blendof 15% degummed and 85% non-degummed silk fibroin was utilized.Approximately 3 grams of powder was mixed with 2 ml of distilled waterand the sample/press was stored in an oven at 90° C. oven for 48 hours.The resulting construct was excellent, with smooth surfaces and lightcolor. Due to its excellent geometric stability, stiffness, andtoughness, the sample was subjected to machinability rests. A TrotecSpeedy 300 Laser Engraver was used to cut some small squares from thesample. Several cutting experiments were run, using varying lasermovement speed and power level (the slower the speed and higher thepower, the more energy is put into the sample being cut). Although laserculling could penetrate the construct and provide the desired geometricshape, the cut edges were burned during the process. The silk fibroinalso created an unpleasant odor during laser cutting. In a secondmachining operation, a drill press was used to create small holesthrough the construct. The drill moved easily through the material,without cracking or chipping the sample. A final machining operation wasperformed using a DeWalt rotozip tool (handheld milling device). Theoperation ran smoothly, although the higher rotational speeds of therotozip tool produced some burning of the machined surfaces.

The fourth sample created with the test protocol utilized a powder blendof 20% degummed and 80% non-degummed silk fibroin. Approximately 3 gramsof powder was mixed with 2 ml of distilled water and the sample presswas stored in an oven at 90° C. oven for 48 hours. The resultingconstruct was excellent, with smooth surfaces and light color. Due toits excellent geometric stability, stiffness, and toughness, this samplewas also used to test machinability.

The fifth sample created with the test protocol utilized a powder blendof 25% degummed and 75% non-degummed silk fibroin. Approximately 3 gramsof powder was mixed with 2 ml of distilled water and the sample/presswas stored in an oven at 90° C. oven for 48 hours. The silk/watermixture had not been evenly distributed in the press, so the resultingconstruct cracked during the release stage of the process. This isattributed to human error; the sample generally exhibited goodmechanical properties otherwise.

New acrylic buffer plate under piston and new sample. To preventdiscoloration of samples due to corrosion of the compaction presspiston, a thin acrylic disk was laser cut and placed between the pistonand sample for subsequent experiments. This design improvement was shownto be effective at preventing further piston corrosion due to exposureto sample moisture and reducing discoloration. A powder blend of 10%degummed and 90% non-degummed silk fibroin was utilized. Approximately 3grams of powder was mixed with 2 ml of distilled water and thesample/press was stored in an oven at 60° C. oven for 24 hours. For thisexperiment, an increase in piston pressure was applied (by lighteningthe hold-down screws described above). Given the rough morphology of thesample, it is assumed that the silk/water combination was poorly mixed.The resulting construct was not homogeneous, although the mechanicalperformance (stiffness and toughness) appeared to be good.

Fabrication of new aluminum compaction press. In order to fabricaterectangular silk constructs for mechanical testing using powdercompaction processing, a new aluminum compaction press was fabricated.The desired geometry was a thin strip with a length-to-width aspectratio of at least 4. The mechanical testing protocol initially involved3-point bend testing. In this test, a sample strip is supportedunderneath by two supports on either end of the sample, while a singleupper support in the center places the sample under a bending load. Tominimize corrosion issues, the entire press was made from aluminum (nosteel parts to generate a brown corrosion product that could discolorthe samples). The new press was designed to produce four samplessimultaneously, including 4 rectangular wells where the silkpowder/binder mixture is placed, “pistons” that transfer pressure fromthe top plate to the samples and hold-down screws.

Powder compaction with patterned die. The regular geometries describedin the prior experiments are useful for a variety of applications. Anadditional range of applications could be envisioned if embeddedfeatures could be created at the surfaces of the silk construct. Toexplore this potential, a special patterned die insert was created fromacrylic (using the single-sample aluminum compaction press). Using animage of an elephant (the Tufts University mascot), the die was createdby laser etching on a Trotec Speedy 300 laser engraver. A powder blendof 25% degummed and 75% non-degummed silk fibroin was utilized.Approximately 3 grams of powder was mixed with 2 ml of distilled waterwhich was added to the well, on top of the acrylic die insert. Thesample and press were stored in an oven at 90° C. oven for 48 hours.This first patterned die was created with low resolution on the laserengraver, so the silk construct did not contain a clean image of theelephant. It was also determined that the powder was not mixedthoroughly before the addition of water, so material in homogeneityresulted (making the elephant image difficult to see).

Powder compaction with patterned die II. This experiment utilized thesame single-sample aluminum compaction press as described above inPowder compaction with patterned die. However, a higher-resolution imageof an elephant was used to create a patterned die. To assist withvisibility, a permanent marker was used to color the area surroundingthe elephant image on the die. A powder blend of 25% degummed and 75%non-degummcd silk fibroin was utilized. Approximately 3 grams of powderwas mixed with 2 ml of distilled water which was added to the well, ontop of the acrylic die insert. The sample press was-stored in an oven at90° C. oven for 48 hours. The resulting construct, shown in FIG. 1A(next to the high-resolution acrylic die insert), had excellentgeometric stability, with a finely detailed version of the elephant.Using a stereomicroscope (FIG. 1B), the laser etcher's raftering andpulsed response are both visible in the silk construct. FIG. 1D showsanother silk construct created using powder compaction and a coin (FIG.1C) as a die insert. The resulting detail replicated in the silk isexcellent.

Samples produced using new multi-sample aluminum compaction press. Aseries of samples were produced using the newly designed and fabricationmulti-sample compaction press. The press has the capability forproducing up to 4 strip constructs simultaneously. A powder blend of 25%degummed and 75% non-degummed silk fibroin was utilized for all samples.Approximately 3 grams of powder was mixed with 2 ml of distilled waterand the sample/press was stored in an oven at 90° C. oven for 48 hours.Because of insufficient filling of the wells with the powder/watermixture, the first samples were thin and cracked in multiple locationsduring heating. Doubling the volume of material per compaction well, asecond set of 4 samples were produced. While improved, these samplesalso exhibited cracking during the heating phase of the process. Usingthe same volume of material, significantly higher pressure was appliedin a third round of sample fabrication. These samples were more robust,although some cracking did occur, resulting in fracturing during sampleremoval from the press.

Example 2. Exemplary Methods Used for Making All-Silk Shoes

The ability to create three-dimensional constructs and/or to performpost-processing operations enables the creation of complex geometries.An exemplary method can relate to the development of an all-silk shoe.This method can form a functional and complete product by bringing manydifferent types of silk-based materials together—foams, fiber/solutioncomposites, the hard constructs described herein, cloth andeletrogelated silk. An exemplary design of the shoe is shown in FIG. 2.In some embodiments, it can support approximately 150 lbs withoutdeforming significantly, and can be a basic high heel design. In otherembodiments, the capacity of the shoe can increase to approximately 300lbs.

Composition of the shoe. The shoe can be composed of several differentforms of silk. Each form has a different processing protocol, and eachmaterial can be optimized for this specific use. The heel and frontplatform of the shoe can be made from a solid form of silk processedusing HFIP (hexafluoro-2-propanol), as can the bottom portion of thesole. The top portion of the sole can be made from silk foam. This foamcan be processed differently to achieve different stiffnesses, and canbe used for both the upper part of the sole, and the padding. The upperpart of the shoe can be made from a composition of silk libers and silksolution; combined to form a silk-silk composite with tailored,directional properties.

Solid HFIP-processed silk for shoe heels, front platforms and bottoms.FIG. 3 outlines the steps to make shoe heels, front platforms, bottomsor any hard parts of the shoe utilizing HFIP-processed silk. To makethis form of silk, there are several steps. First, e.g., silk cocoonsare boiled to separate the proteins (degumming). This degummed silk isthen dried and dissolved using, e.g., lithium bromide. This dissolvedsilk can then be dialyzed with water to remove all traces of lithiumbromide. At this stage, the silk is referred to as silk solution. Thesilk solution can then be freeze dried (lyophilized), at which point itbecomes silk foam. This foam can then be broken up into small, uniformpieces (pulverized), and packed into a mold of a desired shape. HFIP canbe poured on top of the pulverized silk and the moid can be covered.Once this new form of silk is cured, it can be placed in a methanolbath, which washes out the HFIP. The methanol is slowly replaced withwater, until all or most methanol has been removed. After this, thepiece can be dried. Drying can take any time, as long as several months,depending on the size of the molded piece. As the piece dries, it canshrink.

The HFIP processed silk has been previously used to make small, simpleshapes, however it is possible to scale up the production withoutsignificant changes in processing. Mold size and shape can be altered toachieve the desired shape, and a new method of drying the piece can bedeveloped to ensure that even drying takes place in all sections,especially in the heel, since the volume to surface area ratio is largerthan that of the sole.

For use as the sole and heel of a shoe, this new silk material ispreferred to be able to withstand enough force to support a certainamount of weight, as well as the maximum possible impact on the heel dueto walking or running. In one embodiment, the shoe can be designed tohold the weight of a 150 lb person. In another embodiment, a maximumweight of 300 lbs can be allowable. In order to show that this materialis strong enough, the material properties are characterized. One of theessential properties for this application is the mechanical properties:compressive modulus and bending modulus (stiffness). These values can befound by performing various mechanical tests. A finite element model ofthe heel can then be made and the required forces simulated to ensurethe material will not fail in any way under the maximum load. Chemicaltesting can also be done in order to ensure the chemicals used duringprocessing are gone from the final product. Other tests can be done onthe material in order for the product to be sold commercially,flammability and burn tests can be performed, as well as tests forsolvent resistance. Each of these tests is done on a statisticallysignificant sample size.

When the material is shown to have mechanical properties acceptable forthis application, a shoe prototype is constructed. In order to do this,a mold can first be designed and manufactured. The shoe prototype cantake significantly longer to cure and dry than the test samples. Oncemolded, the piece can also be machined, as the shrinkage and warpingthat occurs during drying can distort the original shape. Anyart-recognized methods that can attach the finished pieces made Fromthis material to the rest of the shoe can be used.

Silk foam. The material to be used in the upper part of the sole (thesilk foam) can sesrve as both the support material and the padding inthe sole of the shoe. FIG. 1 outlines the steps for this process. Silkfoam can be made by preparing silk solution as previously described,then freezing it in a mold and lyophilizing it (freeze drying).Depending on the concentration of the silk solution used, the freezingtime, settings of the lyophilizer, and foams of different qualities canbe produced. The properties of the formed silk foam can be tuned tomatch with the padding currently used in shoe manufacture. Compressivemodulus, torsional modulus, re-expansion rate after compression, andmode of failure can be taken into account. These properties can then bereproduced in the silk foam by altering variables in the processing.Post processing steps, such as water annealing and methanol treatments,can also be used to tweak certain properties, which a fleet thecrystallinity of the silk. Other qualities to test in this materialinclude the ease of adhesion and sewabilily of the foam, as well as themechanical properties in a hydrated environment (due to rain or sweat).

Fiber solution composite. The material to be used as the upper portionof the shoe can be composed of silk fibers woven together and coated insilk solution, as prepared by the protocol explained previously. Thereare different alterations to this material, including, e.g., the ratioof fiber to solution, the concentration of the solution, and theformation of the woven fiber. Each of these variables can be optimizedfor the strength and stiffness desired in the final product. FIG. 5shows the steps for this process.

Attachments and connections. Each of the pieces of the shoe can beproduced separately before the assembly of the final product. The heel,upper, platform, padding and lining can be attached to the sole of theshoe. Silk or nonsilk materials can be used to achieve secureattachments. Some of the exemplary attachment methods include, e.g.,silk screws made from solid HFIP-processed silk, embedding fibers intofoams and solid silk forms, and sewing with silk threads.

All patents and other publications identified in the specification andexamples are expressly incorporated herein by reference for allpurposes. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of those documents.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant ailthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedcan be further modified to incorporate features shown in any of theother embodiments disclosed herein.

1. A method comprising: (i) providing a composition comprising silkparticles; and (ii) compacting the composition by application ofpressure into a solid state. 2-39. (canceled)
 40. A method forincreasing compressive strength, clastic modulus, tlcxural stiffness, orshear stiffness of a silk-based material, the method comprising: (i)providing a composition comprising silk particles; and (ii) compactingthe composition by application of pressure into a solid state.